The metabolic activation of the arylbiguanide antimalarials proguanil (PG) and chlorproguanil (CPG) has been investigated in liver microsomes from three human livers. All three microsomal preparations activated the biguanides. The kinetic parameters for PG metabolism to cycloguanil (CG) were Km 21.8, 29.6 and 26.4 microM and Vmax 1.5, 5.9, and 8.2 pmol min-1 mg-1. The values for CPG conversion to chlorcycloguanil (CCG) were Km 12.9, 19.7 and 26.1 microM and Vmax 5.7, 4.8 and 3.6 pmol min-1 mg-1. The metabolic activation of both biguanides was competitively inhibited by the anticonvulsant mephenytoin. Sparteine and tolbutamide had no effect on biguanide metabolism. These data suggest an involvement of the mephenytoin hydroxylase enzyme, which exhibits a genetic polymorphism in man, in the metabolic activation of the biguanide antimalarials.