1. 2,4-Toluenediamine was incubated with liver cytosol from various species, or cytosol from various tissues of the hamster or rabbit, in the presence of [1-14C]acetyl-CoA. N-Acetylation occurred selectively at the p-amino group of 2,4-toluenediamine and to a much lesser extent on the o-amino group. 2. In hamsters and rabbits the highest N-acetyltransferase activity was present in the liver cytosol, followed by kidney intestinal mucosa and lung cytosols. 3. Hamster liver cytosol had the greatest activity followed by liver cytosols from guinea-pig, rabbit, mouse and rat. With human liver cytosol only a trace of an N-acetyl derivative of 2,4-toluenediamine was found while dog liver cytosol showed no activity. 4. N-Acetyltransferase activity was maximal at pH 7-5 in mouse, pH 6-0 in rat and man, and pH 7-0 in rabbit liver cytosols. 5. There was a slight difference in the levels of N-acetyltransferases in males and females; the female mouse had more enzyme activity than the male, but the male rat had more enzyme activity than the female.