Interspecies differences in phenytoin (PHT) metabolism to 5-(4-hydroxyphenyl)-5-phenylhydantoin (HPPH) were examined in human, cat and rat hepatic microsomes in vitro. Rat liver microsomes were 25 and 650 times more efficient at the conversion of PHT to HPPH than human and cat liver microsomes, respectively. Sulphaphenazole (83%) and tolbutamide (TOL) (64%) were the most potent inhibitors of HPPH formation in human liver microsomes, while ciprofloxacin (27%), enoxacin (27%) and TOL (26%) produced the greatest inhibition in cat liver microsomes. TOL was tested for its effect on HPPH formation and gingival overgrowth in cats in vivo. Eight cats received PHT sodium (4 mg/kg/d) and another 8 cats received PHT sodium together with TOL (20 mg/kg/d) for 10 wk. Six cats (75%) in the PHT group and 4 cats (50%) in the PHT & TOL group developed significant gingival overgrowth by the end of the study. However, the extent and incidence of the overgrowth were similar in the 2 groups. There were no significant differences in mean AUC0-10weeks for plasma PHT (552.90 ± 29.36 µg·d/mL [PHT alone] vs. 582.41 ± 24.49 µg·d/mL [PHT & TOL]) and unconjugated HPPH (1016.4 ± 295.5 ng·d/mL [PHT alone] vs. 1174.5 ± 397.2 ng·d/mL [PHT & TOL]) concentrations between the 2 groups of cats. Neither PHT nor HPPH were detectable in the plasma of 8 rats which received PHT (4 mg/kg/d) over a 10-wk period. The rats showed no sign of gingival inflammation (mean gingival index = 0) or gingival overgrowth (mean gingival overgrowth index = 0). Thirty-six adult epileptic patients on chronic PHT therapy were examined; 17 (47%) of the patients demonstrated clinically significant overgrowth. The mean steady-state plasma PHT concentration was comparable to, and the mean plasma unconjugated HPPH concentration 5-fold greater than, that observed in the cats. The results suggest that the rapid metabolism and elimination of PHT and HPPH in the rat may enable it to become more resistant towards developing gingival overgrowth, compared to the cat and man.