A large number of cell lines derived from malignant human neoplasms (21 carcinomas or melanomas and 11 sarcomas or gliomas) and 18 human fibroblast lines were examined for ectopic protein production by RIA. By conservative criteria, 13 malignant lines produced ectopic CG or its β-subunit (0.5-5.6 pmol/mg cell protein). Four of these lines produced ectopic carcinoembryonic antigen (0.04-0.95), while none produced placental lactogen or a-fetoprotein (<0.10). Six malignant lines produced ectopically the common a-subunit of the glycoprotein hormones, and in 2 (ChaGo lung and Chang liver), the secretion rates (391 and 506 pmol/mg cell protein-24 h) were almost 100 times higher than that of any other ectopic protein. Eight malignant lines produced low levels of PRL (0.1-0.57), and 3 lines produced low levels of LH or its β-subunit (0.1-0.26), but neither was secreted. In contrast, only 2 normal fibroblast lines produced CG, and 1 produced PRL-like activity. None of the malignant or fibroblast lines produced GH or FSH or its β- subunit. Moreover, no line produced cortisol, progesterone, 17ahydroxyprogesterone, testosterone, or aldosterone. Estrone and estradiol were found in media from 4 malignant lines (1.1-9.4 pmol/mg protein). These data suggest that ectopic protein production is 1) widely prevalent in malignant cells in culture and rare in normal fibroblast cultures, 2) nonrandom, with large amounts of certain proteins but undetectable amounts of others, and 3) underestimated by criteria of serum concentration alone, since certain lines produce low levels or do not secrete. The multiple enzymes required for stefoidogenesis are not produced together ectopically, but estrogen production by cells in media containing serum may require only a single aromatase or desulfurylase.