Both cervical and vaginal growth are relaxin dependent during rat pregnancy. We recently reported a relaxin-dependent 1.5-fold increase in cervical and vaginal DNA content from midpregnancy until term. This finding indicated that relaxin probably promotes cervical and vaginal growth at least in part by promoting cellular proliferation. The objective of this study was to identify and quantify cells in the cervix and vagina that proliferate during the second half of rat pregnancy in response to relaxin. Primiparous pregnant rats were ovariectomized or sham ovariectomized (group C; n = 8) on day 9 of pregnancy (D9). Ovariectomized rats were then treated with physiological doses of progesterone plus estrogen (n = 7) or progesterone, estrogen, and porcine relaxin (n = 7). Cellular proliferation was determined by continuously administering a low dose of 5-bromo-2'-deoxyuridine (BrdU) via miniature osmotic pump from D9-D22. On D22, cervices and vaginas were collected, fixed in formalin, paraffin embedded, and serially sectioned (4 microm). Adjacent serial sections were either immunostained for BrdU to assess cell proliferation or stained with hematoxylin to determine total cell number. Cell proliferation was evaluated by counting BrdU-positive nuclei and total nuclei in the same area on adjacent sections. Cell counts were determined using computerized digital morphometric analysis at x575. In control rats, nearly 75% of the epithelial cells and 55% of the stromal cells within the cervix at term had proliferated during the second half of pregnancy. The accumulation of approximately half of the new cells was relaxin dependent. Within the cervical stroma, relaxin increased the accumulation of cells associated with blood vessels and also the number of isolated cells (probably fibroblasts). Relaxin did not appear to affect smooth muscle cell proliferation in the cervix. In contrast to the cervix, a minority of vaginal epithelial cells (45%) and stromal cells (20%) proliferated during the second half of pregnancy. Although relaxin appeared to have a tendency to increase the accumulation of new vaginal epithelial and stromal cells, morphometric analysis did not provide support for such an effect. In conclusion, this study demonstrates that relaxin promotes a marked increase in the accumulation of new epithelial cells and stromal cells within the cervix. The relaxin-induced increase in new epithelial and stromal cells probably contributes to relaxin's effects on growth and remodeling of the cervix that are required for rapid and safe delivery.