We categorized established human colon carcinoma cell lines into three biological groups. Our studies were performed on six colorectal cancer lines representing the proposed three groups. Group I consisted of two lines designated LoVo and SW 48; Group II comprised two lines called SW 480 and SW 620; and Group III was represented by lines SW 403 and SW 1116. Group I consisted of the most differentiated cells. This differentiation encompassed morphological markers, gland and signet ring formation, and ciliary development. The outstanding morphological characteristic of Group III was the development of numerous multinucleated giant cells. The range and modal chromosome number increased from Group I to Group III, a change reflected by the higher DNA content of these cells as measured by flow cytometry. Carcinoembryonic antigen synthesis was maximal for Group III and virtually absent for Group II. The number of clonogenic cells decreased from Group I to Group III, while the proportion of nonproliferating cells calculated both by experiments using continuous labeling with tritiated thymidine, and by the primer-available alpha-DNA polymerase index, increased from Group I through Group III. Another important cytokinetic difference was that Group I had an exponential cell cycle stage distribution not seen for the other groups. Cells in Group I were easily propagated in athymic (nude) rats by s.c. injection; cells in Group II injected s.c. grew for about 30 days and then regressed spontaneously. Cells in Group III could only be grown when inoculated intracerebrally. Thus, our studies have now confirmed and extended the hypothesis that cultured human colorectal carcinomas can be separated into at least three groups on the basis of morphological differentiation, chromatin distribution, carcinoembryonic antigen production, cytokinetic properties, and xenograft propagation. Perhaps this classification is just the tip of the iceberg, and future studies will determine the existence of additional groups or subgroups on the basis of other markers. However, at present it appears established that malignant cells with a common histological origin in the gut express their phenotypic potential in a sufficiently discrete manner as to permit their classification into distinct biological groups. Thus, the stage is set for extrapolating this in vitro classification for an in vivo segregation of human colorectal tumors into categories with specific properties and diverse prognosis.