Abstract Incubation of adriamycin resistant Chinese hamster lung cells with low levels of N-ethylmaleimide (NEM) results in a major increase in the cellular accumulation of drug. When resistant cells are prelabeled with [ 32Pi] and thereafter treated with NEM there also occurs a selective superphosphorylation of an 180K plasma membrane glycoprotein (P-180). This phosphorylation reaction occurs at both serine and threonine residues. In similar experiments with drug sensitive cells only minor levels of this protein can be detected. Detailed studies have established that in cells which have reverted to drug sensitivity there is a parallel loss in the presence of phosphorylated P-180. Also in cells which have undergone partial reversion to drug sensitivity there is a correlation between levels of superphosphorylated P-180 and adriamycin resistance. These results provide evidence that adriamycin resistance is dependent on the presence of P-180. The results also suggest that the biological activity of this protein is highly regulated by phosphorylation and that in the superphosphorylated state P-180 is inactive and under these conditions the resistant cell is converted to a drug sensitive phenotype.