Four human colon adenocarcinoma cell lines, SNU-C1, SNU-C4, SNU-C5, and NCI-H716, that are capable of proliferating autonomously in serum-free medium containing no added peptide growth factors were identified. All four cell lines show epidermal growth factor (EGF) receptors (EGFRs), express transforming growth factor alpha (TGF-alpha) messenger RNA, and release anti-TGF-alpha-immunoreactive molecules. The blocking anti-EGFR monoclonal antibody (mAb) 225 blocks autonomous proliferation of SNU-C1 and SNU-C4 cells. In both of these cell lines, the inhibitory effect of mAb 225 is reversible by the addition of EGF, TGF-alpha, or conditioned medium from any of the four cell lines. In contrast, autonomous proliferation of SNU-C5 and NCI-H716 cells is not inhibited by mAb 225 and is not affected by exogenous EGF, TGF-alpha, or conditioned medium. Together, these data confirm the previous finding that anti-EGFR antibodies can inhibit the proliferation of some carcinoma cell lines that coexpress TGF-alpha and EGFR. However, here it is shown that the mechanisms of autonomous proliferation of colon carcinoma cell lines are heterogeneous and not always sensitive to antibody disruption of TGF-alpha/EGFR autocrine interactions.