The occurrence of the neuropeptides peptide histidine methionine (PHM) and vasoactive intestinal peptide (VIP) in the human female genital tract was studied by means of immunochemistry and radioimmunoassay in combination with gel chromatography. In addition, the effect of PHM and VIP on smooth muscle activity was investigated in vitro. The regional distribution of PHM as determined by radioimmunoassay correlated with that of VIP. This finding agreed with the immunohistochemical data, which, in addition, provided evidence for colocalization of the two peptides in nerve fibers. These fibers were most abundant in the vagina and the uterine cervix, where they seemed to innervate blood vessels, smooth muscle, and epithelial cells. The concentrations of immunoreactive PHM and VIP were found to be similar in all areas except in the vagina, where the PHM concentration was fourfold that of VIP. Gel chromatography of vaginal extract revealed a high concentration of a C-terminally extended form of PHM, suggesting differential processing pathways of the VIP precursor. Both PHM and VIP inhibited, in a dose-dependent manner, the smooth muscle activity in strips from the Fallopian tube and the myometrium. Administered in combination, PHM and VIP had an additive effect and displayed the same efficacy as VIP alone, indicating that the peptides act via a common receptor.