L-canavanine, an analog of L-arginine, was examined for toxicity in a normal canine kidney epithelial cell line, Madin-Darby canine kidney (MDCK), and in its chemically transformed derivative MDCK-T1. Under conditions where the analog reversibly arrested growth of MDCK cells, more than 90% of the tumorigenic cells were killed. This selective cytotoxicity was not due to any difference in growth rate (both lines doubled every 24 h). Nor was it caused by the inhibition of protein synthesis or DNA replication, since amino acid deprivation and drugs which inhibited replication directly and indirectly did not kill the transformed cells preferentially. To the contrary, cycloheximide killed MDCK cells preferentially. Although the mechanism for the selective cytotoxicity of canavanine in the tumorigenic cells remains obscure, one clue was afforded by the observation that the analog caused a loss in plating efficiency of the T1 cells prior to their detachment from plates. Selective sensitivity to canavanine may therefore reside in cell surface proteins, which are known to differ both qualitatively and quantitatively between normal and transformed cells. The present results support our previous findings suggesting the possible value of using canavanine as an agent for cancer chemotherapy.