Development of serologic tests to detect recent HIV-1 infection has generated worldwide interest in applying this approach to estimate incidence. We previously devised an IgG-capture BED-EIA (or BED-CEIA) that detects increasing levels of anti-HIV IgG following seroconversion to identify recent infection and to estimate incidence among persons infected with diverse HIV-1 subtypes worldwide. Injection drug users (IDUs; n = 1969) were screened in 1996 for participation in a prospective cohort study. Serum specimens from 594 IDUs were HIV-1 seropositive (30.2%) and were tested with the BED-CEIA. The proportion of recent infections and estimated incidence by different epidemiological risk factors were compared with incidence data measured from the prospective cohort. Of 594 HIV-1-seropositive specimens, 113 (19%) were identified as recent infections. Overall, the estimated annual incidence among persons screened was 17.3%/year (95% CI, 12.8-24.2%/year) compared with 9.0%/year (95% CI, 6.7-11.9%/year) measured from the prospective cohort during the same time period. Estimated incidence was higher among younger aged and unemployed IDUs as well as among those who injected more frequently, confirming previously reported risk factors from this prospective cohort. As persons screened from a cross-sectional sampling probably have higher risk for HIV than selected uninfected individuals who choose to participate and receive risk reduction counseling in a longitudinal cohort study, use of this or other serologic testing strategies to identify populations with high incidence (such as for HIV vaccine trials) may overestimate incidence measured from prospective cohorts.