The mutagenicity of 20 p-phenylenediamine derivatives has been investigated in Salmonella typhimurium. Tests were performed in the presence and in the absence of Aroclor 1254-induced liver S9 fractions derived from male Wistar rats. Among five series of compounds tested, nitro-p-phenylenediamines with substituents at the C6 position (4-amino-3-nitro-6-methylaniline; 4-amino-3-nitro-6-methoxyaniline; 4-amino-3-nitro-6-fluoroaniline; 4-amino-3-nitro-6-chloroaniline; and 4-amino-3-nitro-isopropylaniline) were the most mutagenic. In all cases, the compounds were less mutagenic in the absence of S9 than in its presence, but three of the five compounds (the methoxy, fluoro, and chloro derivatives) were still mutagenic without the metabolic activation system. In contrast to the mutagenicity of the C6-substituted compounds, the mutagenicity of analogues with substituents on the C5 position (4-amino-3-nitro-5-beta-hydroxypropylaniline; 4-amino-3-nitro-5-isopropylaniline; 4-amino-3-nitro-5-methylaniline; and 4-amino-3-nitro-5-beta-hydroxyethylaniline) was abolished or reduced. A dramatic reduction in mutagenic activity was also achieved when two methyl groups, instead of one, were added to 4-amino-3-nitroaniline. For example, 4-amino-3-nitro-5,6-dimethylaniline and 4-amino-3-nitro-2,5-dimethylaniline were only weakly mutagenic, and 4-amino-3-nitro-2,6-dimethylaniline as 4-amino-2-6-dimethylaniline; 4-amino-5,6-dimethylaniline; was nonmutagenic. Monocyclic compounds such as 4-amino-2,6-dimethylaniline; 4-amino-5,6-dimethylaniline; 4-amino-2-methoxy-3,5-dimethylaniline, and 4-amino-2,3,5,6-tetramethylaniline were all nonmutagenic in Salmonella typhimurium. The compound 4-amino-2,5-dimethylaniline was weakly mutagenic or nonmutagenic, whereas 4-amino-2,5-dimethoxyaniline was mutagenic. It appears that the mutagenic activity or inactivity of these compounds depends on both the chemical groups present and their positions in the molecule. In this context, it seems that the presence of the NO2 group and the nature of the substituent groups at the C5 and C6 positions on the benzene ring are crucial factors in determining the mutagenicity of these compounds.