Mice, hamsters and guinea pigs were studied to assess species variation in utilization of pyridoxine-5'-D-glucoside (PN-glucoside), a form of vitamin B-6 found in plants. Animals fed vitamin B-6-deficient or marginally supplemented diets [1 mg pyridoxine (PN)/kg] were given an oral dose of [3H]PN-glucoside plus [14C]PN. Urinary and fecal isotopic excretion was measured over 24 h and the distribution of B-6 vitamins in liver determined at the end of the 24-h period. Intestinal absorption was nearly complete, as very little (< 6%) of each isotope was excreted in the feces. In mice, hamsters and guinea pigs, 31.3, 31.5 and 9.5%, respectively, of urinary 3H was present as intact PN-glucoside. Incorporation into liver was reflected by 3H/14C ratios of hepatic vitamin B-6 as follows: mice, 0.39; hamsters, 0.73; guinea pigs, 1.49 (means for both diets). The intake of dietary vitamin B-6 had little effect on [3H]PN-glucoside metabolism. Guinea pigs displayed greater utilization of PN-glucoside than did mice, hamsters or rats (seen previously), although they may not be the best animal model for the study of PN-glucoside metabolism. Because the bioavailability of PN-glucoside in humans has been estimated to be 58% relative to PN, mice or hamsters, rather than guinea pigs or rats, would be better species for quantitative studies of PN-glucoside bioavailability and associated enzymatic processes.