Antibody-dependent cell-mediated cytotoxicity was used to detect HLA antigens on tissue cultured lymphoblastoid cells (phytohemagglutin blasts and Epstein-Barr virus lines) and transitional cell carcinomas. The results agreed with those obtained on fresh peripheral blood lymphocytes by conventional HLA typing. The same HLA antigens were detected on cells from an individual irrespective of their tissue origin or length of time in vitro. Antibody-dependent cell-mediated cytotoxicity (ADCC) showed that HLA-Cw5 and HLA-Cw8 were cross-reactive. An HLA-Cw5 antiserum that was negative for HLA-Cw8 positive cells in complement-mediated lymphocytotoxicity reacted strongly with HLA-Cw8 donor cells in ADCC. Similarly HLA-Cw8 antibodies were detected in HLA-B14 antisera, which reacted on all HLA-Cw5-positive donor cells. Absorption of sera with HLA-Cw5-positive lymphoid cells removed HLA-Cw5 and HLA-Cw8 specificities but spared HLA-B14. Absorption of HLA-B14 antisera with HLA-B14/Cw8-positive cells removed HLA-Cw5, HLA-Cw8, and HLA-B14 reactivities. Sequential immune precipitation and gel electrophoresis confirmed that HLA-Cw5 and HLA-Cw8 were cross-reactive and that HLA-B14 was physically separable from HLA-Cw8.