In tissue slices of female starlings, binding of [14C]-2-chloro-4-acetotoluidide (CAT) radioactivity to liver proteins was almost five times greater than binding to kidney proteins after 2 h of incubation. Binding to protein of liver slices increased in a log linear fashion with increasing CAT concentrations. Binding to protein of kidney slices also increased with increasing concentrations but not in a log linear fashion. Mixed-function oxidase inhibitors, SKF 525-A and a-naphthoflavone, decreased binding to liver slices but did not affect binding to kidney slices. Anaerobic incubation conditions inhibited binding to both tissues. P-Hydroxymercuribenzoate and sodium cyanide did not affect the binding of radioactivity associated with [ 14C]-CAT to proteins of either liver or kidney slices. Dietbyl maleate increased binding of the radioactivity to proteins of the kidney slices but not to liver slices. Cysteine also increased binding in kidney slices. Binding in liver slices did not increase significantly with cysteine. The cysteine-induced increase in protein binding in kidney slices did not appear to depend on the formation of sulfate from the metabolism of cysteine. There was no sex-dependent difference in starlings as to the binding of radioactivity in either liver or kidney slices. Male chicken kidney slices bound a much higher amount of radioactivity associated with [ "C]-CAT than male starling kidney slices, while the liver slices bound comparable amounts. Male hamster liver slices bound much more radioactivity than did male starling liver slices. However, hamster kidney slices bound much less than did starling kidney slices. Male mouse liver and kidney slices bound much less radioactivity than the comparable tissue slices of male starlings. It was concluded that in vivo metabolism of CAT in starlings plays an important role in covalent binding of [I4C]-CAT radioactivity to kidney proteins and this may be responsible for nephrotoxicity .