Recent evidence indicates that failure of elastic fiber assembly and synthesis is involved in the pathophysiology of pelvic organ prolapse in mice. It has been long been hypothesized that parturition-induced activation of proteases in the vaginal wall and its supportive tissues may contribute to pelvic organ prolapse in women. In this investigation, we determined the expression of matrix metalloproteases with elastase activity (matrix metalloproteinase [MMP] 2, MMP9, and MMP12) and their inhibitors in the vaginal wall of nonpregnant, pregnant, and postpartum mice. Data obtained using mRNA levels and enzyme activity measurements indicate that MMP2, MMP9, and 21- to 24-kDa caseinolytic serine proteases are regulated in vaginal tissues from pregnant and postpartum mice. Although suppressed during pregnancy and the early postpartum time period, MMP2 and MMP9 enzyme activities are increased after 48 h, a time when mRNA levels of protease inhibitors (tissue inhibitor of MMP2 [Timp2], cystatin C [Cst3], and alpha-1 antitrypsin [Serpina1]) are decreased. We conclude that recovery of the vaginal wall from pregnancy and parturition requires increased elastic fiber assembly and synthesis to counteract the marked increase in elastolytic activity of the postpartum vagina.