For quantitative determinations of orcokinin, an indirect, noncompetitive sandwich ELISA was developed. This ELISA is highly specific for orcokinin and the detection limit is 1 fmol. In three astacidean species (Orconectes limosus, Homarus americanus, and Astacus astacus) orcokinin immunoreactivity (OK-IR) was measurable in all parts of the nervous system. Upon normalization to the protein content of the tissue (pmol/mg protein), concentrations were shown to be in the same range in all three species. The distribution of OK-IR in the nervous system is also very similar in the three species. In Orconectes limosus the following values were obtained (in pmol/mg protein): cerebral ganglion 215, optic ganglia in the eyestalk 38, subesophageal ganglion 182. The thoracic ganglia have lower concentrations (35-72) and the abdominal ganglia (AG) 1-5 even lower ones (11-17). In the AG 6 of Orconectes, from which the innervation of the hindgut arises, concentrations are approximately five times higher than in the other AG. In hindgut tissue, relatively high concentrations of 22 pmol/mg were measured, which is in agreement with the demonstrated function of orcokinin as a hindgut excitatory substance. Markedly elevated levels of orcokinin were observed in the AG 6 of Astacus, but not in Homarus. Orcokinin could also be measured consistently and reliably in the hemolymph, where its concentration is approximately 1 x 10(-11) M. These results show that orcokinin may be released into the hemolymph and may act as a hormone, in addition to its role as a locally acting neurotransmitter/modulator.