Techniques presently available for detection of human T-cell lymphotropic virus type III (HTLV-III) antigens and antibodies are laborious or relatively nonsensitive. We adapted anticomplementary immunofluorescence (ACIF) for these purposes. In HTLV-III-infected cells, specific ACIF was demonstrated by a diffuse speckling pattern that often resulted in a peripheral cellular rim of fluorescence. A 97% concordance was demonstrated between the ACIF assay and other sensitive tests for HTLV-III antibody detection (Western blot and membrane immunofluorescence and fixed-cell immunofluorescence tests). The ACIF assay was both more sensitive and more specific when compared with the enzyme-linked immunosorbent assay. For detection of HTLV-III antigens, the ACIF assay appeared to be as sensitive as the reverse transcriptase assay and more sensitive, with less background reactivity, than the conventional immunofluorescence assay. The ACIF assay often detected low levels of HTLV-III antigens within 3 days of infection in vitro, compared with 5 to 7 days with the indirect immunofluorescence assay, and generally paralleled the reverse transcriptase assay. The ACIF assay is a simple, sensitive, and specific assay for detection of HTLV-III-related antigens and antibodies. It should prove useful in the diagnosis of HTLV-III infection, as well as in studies of pathogenesis.