Despite the current use of a standard two-step enzyme immunoassay in the clinical monitoring of the immunosuppressant FK 506, the lack of specificity for the parent drug in this assay renders it unsuitable for drug metabolism studies. An HPLC assay has been developed for studying the metabolism of FK 506 in isolated hepatocytes and microsomal mixtures. This assay allows simultaneous measurement of the parent drug and two of its time dependent metabolites. Metabolism of this drug was studied in intact rat liver cells and rat liver microsomes. We have shown that the metabolites observed are products of phase 1 oxidation reactions. Correlation of the 6 beta-testosterone hydroxylase activity with the FK 506 metabolite (M1) initial formation rate is consistent with the belief that CYP 3A isozymes are involved in FK 506 metabolism in male rats.