Four new cell lines were established from the primary tumors of patients with untreated colorectal adenocarcinoma. Drug sensitivity and characterization of these cell lines was performed. Three of the four cell lines formed colonies in soft agar and all were tumorigenic in nude mice. The cell lines were morphologically similar but had differences in growth characteristics. Two of the cell lines, C18 (CCCL-4) and C29 (CCCL-6), had a longer doubling time compared with C85 (CCCL-1) and C86 (CCCL-2). The C18 and C29 cell lines had chromosome 17 abnormalities and evidence by immunohistochemistry of a mutant p53 and had decreased levels of thymidylate synthase and dihydrofolate reductase proteins, associated with decreased thymidylate synthase catalytic activity in C18 and no detectable activity in C29. Raltitrexed and GW1843U89 showed potent cytotoxic activity and all four cell lines displayed similar cytotoxicity to these folate thymidylate synthase inhibitors. The C18 and C29 cell lines were in general resistant to the other agents tested (methotrexate, 5-fluorouracil, nolatrexed) when compared with the C85 and C86 cell lines. These new cell lines may be useful for the study of colorectal adenocarcinoma and for evaluating new drugs or treatment schedules.