Normal mouse vaginae and uteri regress following ovariectomy, whereas the vagina of mice given five daily injections of 3 micrograms diethylstilbestrol (DES) from the day of birth exhibit ovary (estrogen)-independent persistent stratification and cornification of the epithelium. Zymography indicated expression of four proteinases in both vaginae and uteri of normal mice after ovariectomy. Two proteinases detected in gelatin-containing gel and two others in casein-containing gel proved to be metalloproteinases and serineproteinases, respectively. The two metalloproteinases were identified as gelatinases A and B. Only gelatinase B was intensified 1 d after ovariectomy; however, all four proteinases showed an increase in expression 3 d after ovariectomy. In the uterus, the two gelatinases showed increased expression after ovariectomy. Progelatinase B and serineproteinase II were expressed in the vagina of normal mice at estrus; ovariectomy intensified expression and activation of gelatinases and serineproteinases II in the vagina. Vaginae of mice treated neonatally with DES exhibited a weak expression of proteinases. Ovariectomy changed neither the histology nor the expression of proteinases in these DES-exposed vaginae. Expression of gelatinases was inhibited by estrogen; progesterone stimulated expression and activation of gelatinase B. Serineproteinases found in the vagina and uterus of ovariectomized mice were also inhibited by estrogen but neither was affected by progesterone. These results suggest that gelatinase B and both gelatinases participate in vaginal and uterine regression, respectively, following ovariectomy. Estrogen negatively regulates expression of gelatinases and serineproteinases in the vagina, and of gelatinase A and serineproteinase II in the uterus.