Testing saliva for the detection of human immunodeficiency virus (HIV) antibodies has many potential advantages for epidemiologic surveillance. A commercial ELISA kit and a standardized in-house immunoblot (IB) system were slightly modified to enhance antibody detection in saliva. Frozen saliva specimens from Toronto Sexual Contact Study participants (including sequential saliva specimens collected during seroconversion) were tested as were fresh saliva samples collected from a population of street-based intravenous drug users (IVDUs). HIV antibody results on saliva were compared with HIV serostatus determined by ELISA and IB on serum or dried blood spots. The overall sensitivity was 98.3% (117/119) for the kit and 99.2% (118/119) for IB; the specificity was 100% (429/429). In the IVDU population, compliance in the voluntary submission of specimens increased from 69% agreeing to provide blood samples to 89% agreeing to provide blood, saliva, or both. Saliva specimens can be easily collected under difficult field conditions with minimal training and provide a valuable alternative to testing blood for HIV-seroprevalence studies.