Serum samples totaling 600 were obtained from female prostitutes (300) resident in different hotels in Lagos, Nigeria and nonprostitutes (300) attending different health centers for routine check-up in Lagos, Nigeria. Sera obtained were screened for the prevalence of HIV seropositivity among prostitutes in comparison with nonprostitutes by the enzyme linked immunosorbent assay (ELISA) using the Abbott test kits as recommended by the manufacturers. Positive sera were subjected to confirmatory testing using the Western Blot test-New Lav Blot I and II. Our results revealed that of the 300 prostitutes and nonprostitutes screened, 50 (16.67%) and 6 (2%) were HIV seropositive, respectively, and the difference was statistically significant (P < 0.01). For antibody to HIV-1, 44 (88%) and 5 (83%) of the positive cases recorded among prostitutes and nonprostitutes were seropositive. Four (8%) and one (16.67%) of the positive cases were HIV-2 seropositive for prostitutes and nonprostitutes, respectively. Only 2 (4%) of the positive samples from prostitutes showed reactivities with HIV-1 and HIV-2 (simultaneous infection or dual reactivity). Incidence of the dual reactivity was not recorded among the nonprostitutes screened. This study is expected to provide baseline data on HIV seropositivities among prostitutes and nonprostitutes in Nigeria. The biomedical application of this study is that it will also serve as strong evidence to indicate the high prevalence of HIV seropositivity among prostitutes in order to attract government intervention to step-up control measures.