A protocol for establishment of human colorectal carcinoma cell lines from fresh surgically removed tissues is described. Twelve human colorectal carcinoma cell lines were established from 6 of 18 primary cancers and four of four metastases. Cell lines from concurrent primary tumors and metastases were established from two individual patients. Two primary cancers gave rise to multiple cell lines with differing biological characteristics. Factors contributing to our success appear to be differential selection on the basis of substrate adherence and the timing of passage. The protocol avoids the use of feeder layers or passage through athymic mice. The established cell lines exhibit a range of karyotypes, morphologies, and growth characteristics.