We generated neurotropic herpes simplex type 1 viruses expressing human placental alkaline phosphatase and studied the utility of this enzyme as a marker of infected neurons. The neurotropism of these viruses was assessed by their ability to infect sympathetic preganglionic neurons after adrenal injection in hamsters. The transneuronal transfer of these viruses was examined by their ability to cross the peripheral synapse from the kidney to renal preganglionic neurons or to cross the central synapse from the adrenal gland to the medulla oblongata. Finally, we injected an alkaline phosphatase-expressing herpes simplex virus into the adrenal gland and a beta-galactosidase-expressing herpes simplex virus (US5gal) into the muscular wall of the small intestine to label two neural circuits in one animal and to assess the feasibility of a dual-virus labelling system. The alkaline phosphatase gene was inserted into the glycoprotein J locus or the virus-induced host shut-off locus in the herpes simplex genome to create viruses which replicate (gJHAP HSV or vhsHAP HSV) or into the thymidine kinase locus to generate a virus that does not replicate in neurons in vivo (TK- HAP HSV). Each of the three viruses was retrogradely transported from the adrenal gland of hamsters to sympathetic preganglionic neurons, suggesting that the neurotropism of these viruses was maintained. gJHAP HSV travelled transneuronally from the kidney to sympathorenal preganglionic neurons and from the adrenal gland to neurons in the rostral ventrolateral medulla. Neuronal infection with alkaline phosphatase-expressing virus could be identified using histochemistry but detailed morphology of these neurons was not revealed. However, staining by anti-herpes simplex virus immunoperoxidase demonstrated that they had normal morphology. Identification of two distinct neural circuits in one animal was achieved with our dual-virus labelling system. The nonreplicating TK- HAP HSV was used in combination with US5gal to identify intestinal and adrenal sympathetic preganglionic neurons. The beta-galactosidase-expressing intestinal neurons were labelled bilaterally in the nucleus intermediolateralis, pars principalis, and alkaline phosphatase-expressing adrenal neurons were found ipsilaterally. Some clusters of sympathetic preganglionic neurons in the nucleus intermediolateralis, pars principalis contained mostly intestinal sympathetic preganglionic neurons and a few adrenal sympathetic preganglionic neurons. In other areas, the opposite pattern occurred. About 3-7% of the labelled sympathetic preganglionic neurons were double-labelled by both markers. The distinct and crisp morphology and dendritic processes of neurons stained by beta-galactosidase histochemistry contrasted with the partial staining of neurons by alkaline phosphatase, revealing beta-galactosidase as a better marker of infected neurons. In conclusion, alkaline phosphatase-expressing herpes simplex viruses are yet neurotropic after insertion of this marker enzyme into any of three different loci of the herpes simplex genome. One replicating alkaline phosphatase-expressing virus travelled transneuronally. These alkaline phosphatase-expressing herpes simplex virus can be used together with beta-galactosidase-expressing herpes simplex viruses to determine the target specificity of sympathetic preganglionic neurons controlling visceral organs or can be used to express two different recombinant genes in two targeted neuronal populations. This study suggests that sympathetic preganglionic neurons controlling the intestine and adrenal gland are almost completely distinct.