Cats are highly susceptible to acetaminophen toxicity because of deficient glucuronidation of this drug in vivo. The enzyme kinetic basis for this defect is unknown. Therefore, the kinetic properties of acetaminophen UDP-glucuronosyltransferase (acetaminophen-UGT) were investigated, using hepatic microsomes from cats (N = 4) compared with those of species that are less sensitive to acetaminophen intoxication including dogs (N = 4), humans (N = 4), and six other mammalian species (one liver from each). Gunn rats were also studied, since they express defective UGT family 1 isoenzymes and are also prone to acetaminophen toxicity. Acetaminophen kinetics were biphasic in all instances with distinct high and low affinity components. Km values for the high affinity activity in cat microsomes (0.31 +/- 0.1 mM; mean +/- SEM) were intermediate between those of dogs (0.11 +/- 0.02 mM) and humans (0.60 +/- 0.06 mM) and other species (0.22 to 6.7 mM; range). On the other hand, high affinity Vmax values were over 10-fold less in cat microsomes (0.025 +/- 0.006 nmol/min/mg) than in dogs (0.92 +/- 0.09 nmol/min/mg) and humans (0.27 +/- 0.09 nmol/min/mg); and over 5-fold less compared with microsomes from other species (range 0.13 to 7.63 nmol/min/mg). Gunn rat microsomes showed a similar 10-fold difference in high affinity Vmax values between the homozygous mutant (0.67 nmol/min/mg) and homozygous normal (6.75 nmol/min/mg) animals. These results demonstrate that, relative to a number of other species, cats have remarkably low hepatic levels of a high affinity acetaminophen-UGT. This difference is sufficient enough to explain poor glucuronidation of acetaminophen in vivo and susceptibility to acetaminophen intoxication.