Rat 13762 mammary adenocarcinoma cell clones of differing spontaneous metastatic potentials were tested for their sensitivities to Adriamycin, 5-fluoro-2-′-deoxyuridine (FUdR) and methotrexate in vitro. Cells were treated 4 hours with a single dose of drug, and colony formation was used to assay cell survival. Dose-response curves and survival parameters were calculated for local tumor-derived clones MTC and MTF7 and lung metastasis-derived clone MTLn3. The logarithmic curves were analyzed (slope and y-intercepts) for statistical comparisons. Heterogeneous sensitivities to Adriamycin and FUdR were observed, but there was no difference in the sensitivities of the clones to methotrexate cell killing. We could not find correlations between drug response and clonal origin, passage number or metastatic properties. The sensitivities of the clones to Adriamycin and FUdR changed upon in vitro passage, although drift in Adriamycin sensitivity during growth in culture was not statistically significant. The results demonstrate that (i) clonal heterogeneity exists within the 13762 tumor and its metastases in their inherent responses to chemotherapy agents, and (ii) in the absence of host selective pressures tumor cells can spontaneously and reproducibly drift in their sensitivities to certain chemotherapeutic drugs. © 1983 Taylor & Francis Ltd.