Hybridoma cells have been derived from a fusion between mouse myeloma cells (P3-NSI/1 Ag4) and spleen cells from a mouse immunized with membrane-enriched fractions from the human melanoma cell line Me-43. Of the 26 hybrids obtained, seven secreted antibodies which reacted with the melanoma cell line used for immunization as assessed by an indirect antibody-binding radioimmunoassay. The specificity of the antibodies produced by the seven positive hybrids was further investigated on 16 melanoma cell lines, 15 other tumors, and 14 lymphoblastoid cell lines. The antibodies from four positive hybrids showed a broad reactivity, whereas those from three hybrids reacted exclusively with melanoma cells. The antibodies from two of these three hybrids, α-Mel/5 and α-Mel/14, seem to be directed against common melanoma antigen(s) since they reacted with all (with one exception) of the 16 melanoma cell lines tested, whereas antibodies from the third hybrid, α-Mel/7, reacted only with five of the 16 melanoma lines. Reciprocal binding inhibition tests using [³H]leucine-labeled antibodies showed that α-Mel/5 and α-Mel/14 antibodies were directed against different antigenic determinants.