After injection of 6-thio[8-14C]purine i.p. in rats or rabbits radioactivity was shown to be irreversibly bound to protein of all tissues examined except brain. The binding increased with time after a single injection and increased nonlinearly in response to dose. Binding per milligram of protein was most extensive in intestine, spleen and liver. Binding in liver was primarily to microsomal protein with no binding detected in microsomal nucleic acid. Hepatic microsomal protein binding increased in rats fed a protein-free diet to deplete the hepato-cellular glutathione concentration. Binding to hepatic microsomal protein was decreased by pretreatment with phenobarbital or piperonyl butoxide, but not significantly with allopurinol. 6-Thiopurine at therapeutic doses had no apparent negative effects on either hepatocellular glutathione levels or hepatic microsomal aminopyrine N-demethylase activity during this time period. These observations may be significant regarding reported toxic side effects associated with 6-thiopurine.