The purpose of this open and multicenter trial was to determine the usefulness of antibody detection by enzyme-linked immunosorbent assay (ELISA) in gingival crevicular transudate (GCT), which was collected with an investigational device (Orasure; Epitope, Beaverton, Oreg.), for the diagnosis of human immunodeficiency virus type 1 (HIV-1) infection and to compare it with antibody detection in serum. A total of 1,880 individuals were tested, as follows: 354 HIV-1-infected individuals (111 asymptomatics individuals and 243 individuals with AIDS), 46 individuals with autoimmune diseases (AD), 296 individuals with dental diseases, 42 individuals with other chronic diseases, and 1,142 healthy individuals. Sera from 356 individuals and GCT from 354 individuals were positive for HIV-1 antibodies. There were two false-negative gingival samples, one from an HIV-1-positive asymptomatic individual and one from a patient with AIDS. HIV-1 antibodies were unexpectedly detected in both serum and GCT of two individuals, one with dental disease and one with pulmonary tuberculosis. None of the sera or GCTs from healthy subjects or patients with AD were positive. Compared with the serum assay, the sensitivity, specificity, and positive and negative predictive values of the GCT assay were 99.5, 100, 100, and 99.9%, respectively. Of 355 paired serum-GCT samples that were HIV-1 positive by ELISA and that were tested by Western blot (immunoblot), all were positive for HIV-1 by using the U.S. Public Health Service interpretation criteria, while among gingival samples, 301 were positive, 52 were indeterminate, and 2 were negative. Of 82 negative paired samples selected at random, 80 were negative by Western blotting of serum and GCT and 2 were indeterminate by Western blotting of serum and negative by Western blotting of GCT (a healthy blood donor and a patient with dermatopolymyositis). Testing for HIV-1 antibodies in GCT is a simple and reliable screening procedure in populations with high and low prevalences of infection because of the high sensitivity and specificity of the method, and it offers improved safety for hospital personnel.