The cytosolic proteins and antigens of 11 human colon tumor cell lines were examined with respect to their rate of growth and state of differentiation. Coomassie blue-stained protein analysis of sodium dodecyl sulfate-containing polyacrylamide gels revealed protein bands at Mr 30,000, 31,000, and 58,000, which were characteristic of slower growing and more differentiated cell lines. More rapidly dividing and disdifferentiated colon cell lines lacked the Mr 30,000 and Mr 58,000 bands; instead, they produced a single protein band that ran between the Mr 30,000 and Mr 31,000 positions on the gel. Western transfer analysis of cytoplasmic antigens further subdivided the 11 cell lines into 3 separate categories. Slowly growing and more differentiated lines produced a Mr 52,000 antigen. Intermediate lines, with respect to growth rate and state of differentiation, produced a Mr 38,000 antigen. The rapidly growing and highly disdifferentiated cell lines contained three cytosolic antigens with molecular weights of 37,000, 39,000, and 48,000. These criteria made it possible to classify these 11 human colon tumor tissue culture cell lines into 3 groups which reflect their state of growth activity and degree of differentiation.