BACKGROUND: Reliable methods for estimating the incidence of human immunodeficiency virus (HIV) infection are needed to monitor the epidemic, identify at-risk populations, and evaluate HIV prevention strategies. We used a multifaceted approach to estimate HIV incidence in the HIV Prevention Trials Network (HPTN) 064 study. METHODS: The HPTN 064 study enrolled 2067 HIV-seronegative women and 32 HIV-seropositive women with no prior HIV infection diagnosis. Women were followed for up to 12 months. HIV incidence estimates were based on (1) detection of acute HIV infection, (2) documentation of HIV seroconversion, and (3) detection of recent HIV infection, using a multiassay algorithm (MAA). RESULTS: Two women had acute HIV infection at enrollment, 4 seroconverted, and 2 were identified as recently infected at enrollment using the MAA. The annual HIV incidence estimate based on acute infection at enrollment (2.52% [95% confidence interval {CI}, .17%-9.33%], using a 14-day window period) was higher than the estimate based on seroconversion (0.24% [95% CI, .07%-.62%]; P = .027). Incidence estimates obtained using the MAA at enrollment and at the end of study were 0.25% (95% CI, .03%-.93%) and 0.13% (95% CI, .006%-.76%), respectively. CONCLUSIONS: We detected a high frequency of acute infection at enrollment. Cross-sectional HIV incidence estimates obtained using the MAA were similar to the longitudinal estimate based on HIV seroconversion. Clinical Trials Registration: NCT00995176.