1. Phenacetin O-deethylation catalysed by human liver microcomes has been examined over a substrate concentration range of 2.5 to 700 microM using preparations of eight human liver samples. Michaelis-Menten kinetics described phenacetin oxidation satisfactorily in five of these samples; apparent Km values were in the range of 17.7 to 38.4 microM. 2. In the three other livers a single rectangular hyperbolic relationship did not describe the substrate saturation data adequately; analyses in these three cases requiring two classes of catalytic site. The apparent Km value for the higher affinity class of site in these three samples was within the range quoted above, but limitations imposed by assay sensitivity and phenacetin solubility obviated accurate characterization of the lower affinity class. 3. Estimates of Vmax for the high affinity class of site in the eight livers varied eleven-fold and there was no correlation between either Km or Vmax and microsomal cytochrome P-450 specific content, NADPH cytochrome c (P-450) reductase specific activity or ethylmorphine demethylase activity. 4. Propranolol was a potent competitive inhibitor of phenacetin deethylation with apparent Ki values of 2 to 7 microM describing its effect on the higher affinity class of activity. Propranolol was also an inhibitor of the lower affinity phenacetin deethylase identified in three of the livers, however the mechanism of inhibition could not be characterized. 5. These data suggest the possibility that propranolol oxidation may be mediated in part, by one or more human liver cytochrome P-450 species catalyzing phenacetin oxidation.