The relative distribution of B-6 vitamers, separated by reverse-phase liquid chromatography, was examined in human milk during a 24-h period after supplementation with 2.5 or 15 mg pyridoxine hydrochloride. Consistently, pyridoxal (PL) was the predominate vitamer and the most responsive to vitamin B-6 intake. During 3-8 h after supplement ingestion, PL, pyridoxal phosphate, and pyridoxamine concentrations were significantly higher than at other times examined. In the first two periods after supplementation, PL as a percentage of total vitamin B-6 was slightly but significantly higher in milk from the group supplemented with 15 mg than from the group supplemented with 2.5 mg. With the exception of PL, the distribution of B-6 vitamers, expressed as percent of total vitamin B-6, was similar for the two supplemented groups at all times examined. Percentage PL of total vitamin B-6 in milk was approximately 25% lower in unsupplemented than in supplemented women.