Conditioned media collected under serum-free conditions over 24 to 48 h from 18 human colon adenocarcinoma cell lines were analyzed for transforming growth factor, types α and β (TGF-α and -β), and platelet-derived growth factor in assays for anchorage-independent growth and radioreceptor competition. Detectable levels of TGF-α, TGF-β, and platelet-derived growth factor were produced by 17, 16, and 6 cell lines, respectively. Three liters of conditioned medium from highly tumorigenic (HT-29, DLD-1, and SW620) and nontumorigenic (SKCO-1) colon cell lines and from nonneoplastic rat kidney (NRK-52E) and small intestinal (IEC-6) epithelial cells were purified by high-performance liquid chromatography and assayed for TGF-α- and TGF-β-like activity. The highly tumorigenic colon cell lines produced 10- to 45-fold (soft agar), 19- to 90-fold (radioreceptor), and 4- to 35-fold (radioimmunoassay) more TGF-α activity compared to the nonneoplastic rat intestinal (IEC) epithelial cells. NRK-52E did not produce detectable TGF-α activity. Radio-immunoassay analysis of peak fractions revealed only TGF-α immuno-reactivity; epidermal growth factor was not detected. Levels of TGF-β-like material in the colon carcinoma populations were comparable (HT-29) or elevated (DLD-1, SW620) only 3- to 4-fold (soft agar) or 1- to 3-fold (radioreceptor binding) compared to IEC cells or NRK-52E. Growth factor production is an ubiquitous property of colon carcinoma cell lines maintained in vitro and is consistent with this class of molecule, playing a contributory role in regulating cell growth.