A malignant B16 melanoma parental cell line, four unselected clones derived from this line which possess varying lung colonization potentials (clones 9 and 14 > parental B16 > clones 12 and 15), four variant cell lines selected in vivo for increased ability to colonize preferentially lungs (B16-F10 > B16-F1, parental B16), brain (B16-B10n > B16-F1, parental B16), or ovaries (B16-O10 > B16-F1, parental B16), and a line selected in vitro for resistance to lymphocyte cytotoxicity (B16-F10^Lr-6) have been examined in culture for their susceptibilities to growth inhibition by retinoic acid. The proliferation of all clones and cell lines was inhibited in the presence of noncytotoxic concentrations of retinoic acid (10⁻⁵ and 10⁻⁶ m); however, the extent of inhibition varied significantly. The parental B16 line and parental clone 14, as well as the in vivo-selected variant cell lines B16-F1 and B16-B10n, were extremely sensitive to retinoic acid, and their growth was inhibited by 75 to 90% after a 5-day incubation in the presence of 10⁻⁵ m retinoic acid. Under similar conditions, the growth of parental clones 9, 12, and 15 and of selected cell lines B16-F10, B16-F10^Lr-6, and B16-O10 was inhibited only moderately (40 to 60%). A more striking difference in sensitivity was observed when cells were exposed to a low retinoic acid concentration (10⁻⁹ m). While the parental B16 line and B16-F1 were inhibited by nearly 40%, parental clones 12 and 14 and selected cell line B16-F10^Lr-6 were inhibited by about 20%, and parental clones 9 and 15 and lines B16-B10n, B16-O10, and B16-F10 were not significantly affected (0 to 10% inhibition). These results demonstrate that the parental B16 melanoma line is heterogeneous with respect to sensitivity to retinoic acid and includes some cell variants with reduced susceptibility to the drug.