The aglycone methylazoxymethanol of the naturally occurring carcinogenic glucoside, cycasin, has previously been shown to be mutagenic, but cycasin per se has not. In this work, cycasin was demonstrated to be mutagenic using a modification of the Ames Salmonella test in which it was preincubated with beta-glucosidase and the tester strain in liquid medium. The mutagenicity of cycasin to six histine-depedent Salmonella strains varied considerably with strain HisG46 being the most susceptible. Methylazoxymethyl-beta-D-glucosiduronic acid, which also is nonmutagenic per se, similarly became mutagenic when preincubated with beta-glucuronidase. Methylazoxymethyl acetate, which is slightly mutagenic by the Ames standard pour plate method, became highly mutagenic on preincubation. The mutagenicity of free methylazoxymethanol was confirmed, and a linear dose-response relationship was observed. The common conditions required for activation of nonmutagenic methylazoxymethanol conjugates, the glucoside cycasin and methylazoxymethyl-beta-D-glucosiduronic acid, are 90-min preincubation at 30 degrees, pH 6.5, with an appropriate hydrolase and Salmonella typhimurium HisG46.