A rat fibroblast cell line (Rat-1) carrying a temperature-sensitive mutation of v-src was used to determine whether inducible cellular transformation altered the ability of cells to amplify the p-glycoprotein gene in response to colchicine selection. Transformed and nontransformed Rat-1 fibroblasts selected under 4 times the LD 50 generated the same number of colchicine-resistant colonies. We next examined colchicine-resistant colonies derived from transformed cells and compared them to colchicine-resistant colonies derived from nontransformed cells. When Rat-1 cells were selected at 35°C (transforming temperature), 7 out of 7 clones exhibited a 3- to 5-fold p-glycoprotein gene amplification. These results contrasted to those found at the nontransforming temperature (40°C); none of the 8 colchicine-resistant clones examined had amplified the p-glycoprotein gene. Thus in Rat-1 cells carrying a temperature-sensitive v-src gene, p-glycoprotein gene amplification was observed at a high frequency only in transformed clones selected at the temperature permissive for v-src activity.