3,5-Dichlorobenzidine and 3,5,3'-trichlorobenzidine were prepared by the reaction of 2,6-dichloronitrosobenzene with aniline and 2-chloroaniline, respectively, to give the appropriate substituted azobenzene; reduction and rearrangement gave the desired benzidine derivatives. The products were purified and characterized by mass spectrometry and [1H] nuclear magnetic resonance spectroscopy. The mutagenic activities of the chlorinated benzidines were determined, using the Ames tester strain TA98. 3,5,3'-Trichlorobenzidine is the most potent benzidine derivative yet reported. The activities of five chlorinated benzidines are compared, and rules for the effect of substitution on activity are discussed.