The study of interventions to prevent HIV transmission requires access to populations with a high rate of HIV transmission. We estimated HIV incidence among heterosexual males and females who were seen at an HIV testing site in Rio de Janeiro, Brazil. Stored sera from individuals who visited the site between March and December 1998 were analyzed using the sensitive/less sensitive (S/LS) assay and a chart abstraction was performed. During the study period, 6353 serum samples were tested. Of those tested, 1203 were found to be HIV-seropositive or indeterminate, of which 1050 (87%) remained available for further testing. In addition, 84 serum samples, representing 63 adults, were found to produce results suggesting early HIV infection. Of these, 14 were heterosexual and female (median age, 38 years), and 19 were heterosexual and male (median age, 25 years). The estimated HIV seroincidence was 1.9 (95% confidence limits (CL), 0.9%-3.9%) and 2.8 (95% CL, 1.4%-5.3%) per 100 person-years among heterosexual women and men, respectively. A survey on willingness to participate in future placebo-controlled HIV vaccine trials in this population indicated that 54.5% and 53.9% of heterosexual women and men, respectively, indicated that they would definitely be willing to participate. We have identified a heterosexual population in Rio de Janeiro with a high rate of HIV transmission willing to participate in placebo-controlled vaccine trials. This study demonstrates the usefulness of the newly described S/LS assay, which allows one to estimate HIV incidence from single serum specimens.