A monoclonal antibody (MAb 76) was produced by immunizing mice with a soluble cytoplasmic protein fraction from a human adenocarcinoma of the colon. MAb 76 showed specific immunoreactivity against a 76 kDa protein in immunoblot studies using total colon tumor cytosol proteins. Immunoprecipitation of phosphorylated cytosolic protein products with MAb 76 and subsequent analysis on SDS containing polyacrylamide gels revealed a single 38 kDa band, indicating that the 76 kDa antigen is associated with a 38 kDa phosphoprotein species. Indirect immunofluorescence analysis of primary tumor specimens and human colon tumor cell lines showed positive immunoreactivity with 6/7 human colon adenocarcinoma tissues and 15/18 human colon tumor cell lines. MAb 76 was unreactive with normal colon, liver and lung specimens from human, mouse and hamster. The epitope-bearing monomer detected by MAb 76 is immunologically conserved in a high percentage of colon tumor cells and tissues and may represent a cellular product that is characteristic of the transformed colon cell phenotype.