AbstractOur previous reports of major sex differences in the substance P‐immunoreactive (SPir) innervation of the medial posterior divisions of the bed nucleus of the stria terminalis (BST) and medial nucleus of the amygdala in rats raised the question of the hormonal regulation of this innervation. We now report the results of two experiments which examined the effects of castration of adult males on the SPir innervation of these regions. In experiment 2 we asked whether castration might also alter the cytoarchitecture of these regions.In experiment 1 three groups; sham operated (Sham), castrated (C) and castrated plus testosterone (C + T) were examined at each of the three survival periods (2, 4 and 8 weeks) post castration. Animals of the C + T groups each received a 45 mm silastic implant of testosterone sc at the time of castration to maintain testosterone levels postoperatively. Castration produced a consistent and highly significant decrease in the area of dense SPir fiber staining in the posterior medial amygdala which became greater with increasing survival. By 8 weeks the area of staining was 42% smaller in group C as compared to the matched sham‐operated group. Smaller decreases were seen in the size of the dense field of SPir fibers in the posterior part of the dorsomedial BST. Testosterone implants maintained the size of the SPir fields of fibers in both the medial amygdala and BST, as the areas of staining in the C + T groups were not significantly different from those in the Sham groups at any of the 3 survival times.In experiment 2 we measured the area and optical density of SPir fiber staining in the medial amygdala and medial BST at 8 weeks post‐castration. In addition, we measured the size of the cell groups within these regions using cresyl‐stained sections. As in experiment 1, at 8 wks following castration there was a marked decrease in the area of dense SPir staining in both the BST and medial amygdala. The sizes of the dense fields of fibers were reduced by approximately 23% in the BST and by 40% in the posterior medial amygdala. Castration also significantly reduced the optical density of staining within the medial amygdala. The major finding of experiment 2 is that castration affects the cytoarchitecture as well as the SPir staining in these areas. In the BST, the cell group BSTMPM receives most of the dense SPir innervation. Gonadectomy reduced the size of BSTMPM by approximately 28%. In the amygdala, the cell group MePD receives most of the dense SPir innervation. Gonadectomy reduced the size of MePD by approximately 27%, while its neighbour MePV was reduced by a similar degree (26%). These atrophic changes are at least somewhat specific, as other features such as brain weight, the overall size of the forebrain as estimated from whole coronal sections, and the size of the suprachiasmatic nucleus were unchanged. The atrophic changes in the cytoarchitecture of the posterior medial amygdala and BST suggest that the changes in SPir staining seen in experiments 1 and 2 may be secondary to structural atrophy, including reduced axonal and dendritic branching, of hormone responsive SP‐containing neurons. The time course of the response to castration demonstrated in experiment 1 suggests that these changes in SP‐containing neurons are relevant to the gradual decline in male sexual behavior which follows