The absorption of vitamin B-6 from the gastrointestinal tract of the chicken (Gallus domesticus) was studied by using ligated segments in vivo, everted jejunal sacs in vitro, and in intact birds. [3H]Pyridoxine hydrochloride ([3H]PN . HCl) was absorbed from all sections of the small intestine, from the cecum and the crop, although absorption from the latter two segments was minimal. Absorption was independent of fasting and the vitamin B-6 concentration in the rearing diet. Concentration-dependence was demonstrated for absorption from ligated jejunal segments (24 microM-24 mM) and for 4-min uptake of [3H]PN . HCl by everted sacs (0.01-10,000 microM). Unidirectional flux of 2 microM PN . HCl was reduced by ouabain, iodoacetate and Na+-free media but not by 4-deoxy PN . HCl, D- and DL-penicillamine, anoxia or glucose-free media. Absorption of vitamin B-6 from the lumen to the intestinal epithelium of the chicken occurs by simple diffusion. Free, added vitamin B-6 was almost completely absorbed. In contrast, vitamin B-6 in food ingredients became available only as digestion proceeded and was in no case completely available. The vitamin B-6 concentrations in the contents of the cecum, lower ileum and rectum were similar. Incomplete absorption of dietary vitamin B-6 could explain the presence of the vitamin in the cecum. An enterohepatic route accounting for the recycling of approximately 1% of normal daily vitamin B-6 intake was identified, and the vitamin B-6 concentration in bile increased with vitamin B-6 in the diet. Pyridoxal, pyridoxamine (and their phosphate esters), pyridoxine, and 4-pyridoxic acid were measured in blood, but only the pyridoxal concentration in blood responded noticeably to increases in dietary vitamin B-6.