The mechanisms of polymorphic drug hydroxylation of debrisoquine, sparteine and related drugs in vivo have been investigated using Cyt P-450 preparations of inbred rat strains as an in vitro model of the poor and extensive metabolizer phenotypes found in various rat strains and in man. Optical difference spectroscopy with debrisoquine, sparteine, phenformin and three other drugs (selected test compounds with proven or suspected hydroxylation polymorphisms in man) exhibited Type 1 binding in normal Sprague-Dawley, Fischer and Lewis Cyt P-450, whereas no Type I drug binding was found in the hydroxylation deficient DA rat liver Cyt P-450. Cyt P-450 content and Type II drug binding of metiamide was the same in normal and hydroxylation deficient rat liver microsomes. The pronounced Type I drug binding in extensive hydroxylation Cyt P-450 and the defective Type I binding in DA Cyt P-450 in vitro, therefore, closely parallels the polymorphic hydroxylation pattern of these test drugs found in the four rat strains studied in vivo. Consequently, missing binding properties of Cyt P-450 or of its micro-environment might represent the enzymatic defect underlying the genetically determined hydroxylation deficiency of polymorphically metabolized drugs in the poor metabolizer phenotype in the DA rat and, by inference, in man.