Objectives: Observational data suggest depot medroxyprogesterone acetate (DMPA) and pregnancy increase HIV susceptibility. We compared proportions of immune and HIV target cells in the female reproductive tract from women using hormonal or intrauterine contraception, no hormonal or intrauterine contraception, and pregnancy. Methods: We collected samples from the cervical transformation zone, endocervix, endometrium and blood from pregnant women (n=5), and women using DMPA (n=5), oral contraception containing levonorgestrel (LNG-OC; n=15), 52 mg LNG intrauterine device (LNG-IUD; n=14), copper IUD (Cu-IUD; n=15), and no hormones or IUD (controls; n=15). Biopsies were timed to peak progestin levels. We performed immunophenotyping on fresh cells isolated from each tissue site to assess the distribution of immune cells including CD4+T cells, dendritic cells, Langerhans cells, and macrophages. Results: In the endocervix, immune cell distributions were significantly different when comparing LNG-OC, LNG-IUD, Cu-IUD and controls (p=0.005), but not when comparing DMPA, pregnant and control samples. In the endometrium, IUD use significantly affected the distribution of immune cells (p=0.04), but LNG use did not. In blood, the distribution of cells in DMPA users was statistically different from pregnant and control samples (p=0.05), but not among LNG-OC, LNG-IUD, Cu-IUD, and control samples. No statistical differences between groups were found when comparing cell distributions in the cervical transformation zone. Conclusions: Studies assessing contraceptive effects on HIV susceptibility should evaluate multiple female reproductive tract compartments, and consider including a pregnant comparison group given that in many high HIV prevalence regions, contraceptive methods are limited and unintended pregnancies are frequent.