Objective: To identify the effects of depomedroxyprogesterone acetate (DMPA) on vaginal microbial flora and epithelium. Methods: Women who desired DMPA for contraception were evaluated before and at 3 and 6 months after initiation of 150-mg DMPA injections every 3 months. At each visit, we assessed genital symptoms, vaginal signs, vaginal microflora, and histopathology by vaginal biopsies. Results: Among 38 women observed for 6 months, there was significant reduction in mean serum estradiol level (99.9 +/- 9.3 pg/mL to 26.6 +/- 1.6 pg/mL, P < .001). The number of subjects with any Lactobacillus did not change, but the number with hydrogen peroxide (H2O2)-positive Lactobacillus decreased from 20% before to 12% after 6 months of DMPA (P = .005). The log concentration in colony-forming units per milliliter of vaginal fluid of H2O2-positive Lactobacillus decreased in a linear manner from 4.0 +/- 0.6 at baseline to 2.5 +/- 0.6 after 6 months of DMPA use (P = .006). The mean number of cell layers in the epithelium was reduced slightly from 28.1 +/- 0.7 to 25.9 +/- 0.9 (P = .05), epithelial thickness decreased from 1.02 +/- 0.04 mm to 0.89 +/- 0.05 mm (P = .005), and the glycogen-positive thickness decreased from 0.81 +/- 0.04 mm at baseline to 0.66 +/- 0.05 after 6 months of DMPA use (P = .005). Conclusion: Depomedroxyprogesterone acetate produced a systemic hypoestrogenic state associated with decreased H2O2-positive Lactobacillus colonization and slight thinning of the glycogen vaginal epithelial layer. Such changes possibly compromise the vaginal barrier to infection. (Obstet Gynecol 2000;96:431-9. (C) 2000 by The American College of Obstetricians and Gynecologists.).