Three experiments were performed to determine whether renal afferent pathways were activated by the diuretic drug, furosemide. It was hypothesized that activated neurons of the renal afferent pathway would express the protein product Fos of the c-fos immediate early gene and be identified by immunocytochemical staining for Fos in the cell nucleus. In the first two experiments, rats were injected with either furosemide (5 mg) or vehicle solution (sterile isotonic saline) and sacrificed either 1.75 h (short-survival experiment) or 3.5 h (long-survival experiment) after injection. In both experiments, the furosemide-treated rats had significantly more Fos-positive cell nuclei than vehicle-treated rats in the subfornical organ (SFO), organum vasculosum lamina terminalis (OVLT), supraoptic nuclei (SON), and magnocellular region of the paraventricular nuclei (PVN) - areas previously shown to be activated by hypovolemia or peripheral angiotensin. In the short-survival experiment, the furosemide-treated rats had more Fos-positive cell nuclei in the nucleus of the solitary tract (NTS) and in the dorsal horn of the spinal cord at spinal levels T(11), T(12), and T(13). In contrast, furosemide treatment did not produce more Fos-positive cell nuclei in the NTS and dorsal horn of the spinal cord in the long-survival experiment. These results suggest that the activation of the SFO, OVLT, SON and PVN may be via a different mechanism than that of NTS or spinal cord dorsal horn. Based upon our previous work, we hypothesized that the NTS and spinal cord dorsal horn labeling was due to activation of sympathetic afferents originating in the kidney and labeling in forebrain structures was due to stimulation by angiotensin generated by renal renin release. To test this hypothesis, a third experiment was devised that was identical to the short-survival experiment, except that all rats had bilateral renal denervation surgery 1 week previously. In this experiment, furosemide administration increased the number of Fos-positive cells in the SFO, OVLT, SON and PVN, but not in the caudal thoracic spinal cord or NTS. These results together with the results of first two experiments lend support to our hypothesis that furosemide-induced neuronal activation in the thoracic spinal cord and NTS is due to activation of second- and/or third-order neurons of a renal sympathetic afferent pathway. Furosemide-induced activation in the SFO, OVLT, SON and PVN does not depend on renal innervation. It is hypothesized that activation in these forebrain regions depends on the action of angiotensin II that is generated after furosemide treatment. Our results indicate that both a hormonal pathway and a renal sympathetic afferent pathway conduct information from the kidney to the central nervous system (CNS) after furosemide treatment.