BACKGROUND Antiretroviral therapy (ART) may influence the biological, social and behavioral determinants of pregnancy in HIV-infected women. However, there are limited longitudinal data on the reproductive intentions and outcomes among women on ART in Africa. METHODOLOGY /PRINCIPAL FINDINGS: Using a prospective cohort design, we analyzed trends in desire for children and predictors of pregnancy among a cohort of 733 HIV-infected women in rural Uganda who initiated ART between May 2003 and May 2004 and were followed up in their homes until June 2006. Women answered in-depth social and behavioral questionnaires administered every quarter in year 1 after initiating ART, and every 6 to 12 months thereafter. Use of family planning methods was assessed at 18 and 24 months after starting ART. We tested for non-constant pregnancy incidence by using a shape parameter test from the Weibull distribution. We modeled repeated measurements of all variables related to the women's desire for children over time using a generalized estimating equation (GEE) extension to the logistic regression model. Risk factors for pregnancy were examined using Cox proportional hazards model. 711 women eligible for the study were followed-up for a median time of 2.4 years after starting ART. During this time, less than 7% of women reported wanting more children at any time point yet 120 (16.9%) women experienced 140 pregnancies and pregnancy incidence increased from 3.46 per 100 women-years (WY) in the first quarter to 9.5 per 100 WY at 24 months (p<0.0001). This was paralleled by an increase in the proportion of women reporting sexual activity in the past 3 months, from 24.4% at baseline to 32.5% over 24 months of follow-up (p = 0.001). Only 14% of women used permanent or semi-permanent family planning methods by their second year on ART. In the multivariate model, younger age (HR = 2.71 per 10-year decrease, 95% CI: 2.95-3.78), having a BMI>18.5 (HR = 1.09, CI: 1.01-1.18) and not having used condoms consistently in the last 3 months (HR = 1.79, CI: 1.02-3.13) were independently associated with pregnancy. CONCLUSION/SIGNIFICANCE Women on ART and their partners should be consistently counseled on the effects of ART in restoring fertility, and offered regularly free and comprehensive family planning services as part of their standard package of care.