The human colon carcinoma cell line DLD-1, established from tumor tissue obtained from a 45 year old white man with an adenocarcinoma of the sigmoid colon, was studied from the perspective of tumor heterogeneity. The karyotype and morphology of cells from an early passage DLD-1 culture, as well as the histologic features of both the original tumor and neoplasms produced by inoculation of athymic nude mice with DLD-1 cells, indicated that both the DLD-1 cell line and the original tumor were heterogeneous. Two clones were isolated from the DLD-1 line; they differed in their morphology, karyotype, and cloning efficiency in soft agar. Furthermore, when cells from each clone were injected into athymic mice, histologically distinct tumors were produced. Various analyses showed that the two cloned lines were representative of the two subpopulations predominantly responsible for the heterogeneity of the original neoplasm. In vitro drug screening results demonstrated that the two cloned lines have differential sensitivities to chemotherapeutic agents. The parent DLD-1 human colon carcinoma cell line and its two cloned subpopulations provide material for the study of various aspects and implications of human cancer cell heterogeneity.