Serum and saliva from 195 known human immunodeficiency virus (HIV)-seropositive patients and 198 military personnel undergoing annual HIV serologic testing were evaluated in a prospective, blinded fashion for anti-HIV-1 antibodies. Oral specimens, collected with a device designed to concentrate oral mucosal transudate from whole saliva, were tested by a modified ELISA and by Western blot. Serum was tested in a standard manner. All 195 HIV-1-seropositive subjects had detectable anti-HIV-1 antibodies in their saliva by ELISA; 190 saliva samples were positive by Western blot and 5 were indeterminate. None of the 198 military personnel were positive by ELISA of serum or oral fluid. The sensitivity, specificity, and positive and negative predictive values for ELISA of saliva were each 100%. The serologic testing of oral mucosal transudate appears to be a simple, safe, sensitive, and specific method for detecting anti-HIV-1 antibodies.