The structural basis for direct mutagenicity of acridines was studied by testing 50 different analogs in the Ames Salmonella tester strains without the addition of mammalian activating enzymes. These experiments showed that the single most effective substituent for frameshift mutagenesis in strain TA1537 is an amino group at the "9" position, while an amino group at either the "3" or "1" position is less effective. Other substitutions at the "9" position demonstrate decreased frameshifting activity compared to 9-aminoacridine. Furthermore, all substituents in combination with the amino group of 9-aminoacridine also decrease frameshifting activity, except for the addition of another amino group at the "1" position or a methyl at the ring nitrogen. Nitro substituents at the "1" and "3" positions enhance 9-aminoacridine toxocity. All nitro substituents decrease typical acridine-frameshift mutagenesis for strain TA1537, but they induce mutagenic activity either in the other type of frameshift strain, TA1538, or in the base-pair substitution strain TA1535. These studies have provided important structure-function relationships for acridine mutagenicity and toxicity in Salmonella. Consequently, this biological system has provided a sensitive means for determining the structural requirements for mutagenic mechanisms.