The conversion of thyroxine (T4) to triiodothyronine (T3) was studied in homogenates and subcellular fractions of 10 human liver specimens obtained postmortem. Preliminary studies indicated that T4 5'-deiodinase activity did not decline in rat liver kept at 5 degrees C for 6 and 24 hr after death. All human liver homogenates but one catalyzed T3 production, although the quantity of T3 produced varied greatly, from 8-fold in the absence of dithiothreitol (DTT) to 100-fold in its presence. The wide variation in activity found may reflect either postmortem loss or premortem decline in enzyme activity due to unrecognized nonthyroidal illness. The amount of T3 produced was dependent on substrate availability, protein concentration, time, pH and temperature, and enzyme activity was greatest in the microsomal fraction. T3 production was stimulated by DTT and inhibited by propylthiouracil (PTU). Thus, human liver T4 5'-deiodinase has properties very similar to the same enzyme in rat liver. These data suggest that results of studies of the effects of nonthyroidal illnesses and drugs on T4 5'-deiodinase activity in rat liver may be extrapolated to humans.