The hormonal regulation of sex differences in electrocommunicatory behavior and brain substance P-like immunoreactivity (SPI-ir) were examined in the weakly electric fish, Apteronotus leptorhynchus. This animal modulates its electric organ discharge (EOD) to produce discrete electric social signals (chirps), which function in aggressive and reproductive displays. Males readily chirp in response to electrosensory stimuli that mimic the presence of a conspecific; females also chirp in response to such stimuli, but do so at much lower rates than males. We have recently demonstrated that androgen treatment enhances chirping behavior in females and may also lead to a change in chirp quality or structure. In this study, we quantified androgen- induced changes in chirp structure and simultaneously examined whether androgens alter the sexually dimorphic pattern of SPI-ir in a brain region (prepacemaker nucleus, PPn) known to control chirping. Our results demonstrate that, in females, androgens cause both the induction of chirping and an alteration of chirp structure; chirps recorded from androgen-implanted females had longer durations and more dramatic frequency and amplitude modulations compared to controls, and appear similar to those reported to be produced during spawning. Moreover, androgen-induced changes in chirping are correlated with increased expression of SPI-ir within specific brain nuclei of females. These changes may underly behavioral changes in chirping, since treated females showed a male-like pattern of SPI-ir in the PPn. However, alterations in SPI-ir were not restricted to the PPn, but also occurred in diencephalic regions related to pituitary function and reproductive behavior. The results suggest that androgens modulate chirping activity and cause both specific and wide-spread changes in SPI-ir that may relate to a functional system that interrelates pituitary function, reproductive behavior, and chirping.