Study Objective: To evaluate performance characteristics of sequential enzyme immunoassay (EIA) and Western blot human immunodeficiency virus type 1 (HIV-1) antibody testing in a low-risk population. Design: Three-year prospective study of a selected sample from a community-based population. Setting: Two blood collection facilities in Minnesota. Population: Minnesota blood donors. Results: During the study period, 630,190 units of blood (donations) from an estimated 290,110 Minnesota-resident donors were screened for HIV-1 antibody. Seventeen Minnesota-resident donors were identified as positive for HIV-1 antibody. Sixteen donors were available for follow-up HIV-1 culture: all were culture positive. The other donor, who was not available for follow-up culture, was likely infected with HIV-1 based on a history of high-risk behavior and positive serologic findings for hepatitis B surface antigen. Using 95% binomial confidence intervals, performance characteristics for sequential EIA and Western blot HIV-1 antibody serology were as follows: false-positive rate by number of donations, 0% to 0.0006%; specificity by number of donations, 99.9994% to 100%; predictive value of a positive test, 81% to 100%. Conclusions: In this low-risk population, the false-positive rate of serologic tests for HIV-1 antibody, using HIV-1 culture as the definitive standard for infection status, was extremely low and test specificity was extremely high.