The availability of vitamin B6 and pantothenate in an average American diet was assessed healthy male volunteers. The subjects received two types of diets, both nutritionally equivalent to the average American diet: period 1 (35 days), semipurified formula diet (low in both vitamins) with daily supplements of 1.1 mg pyridoxine and 8.2 mg pantothenate; period 2 (35 days), natural food sources, providing 2.3 mg vitamin B6 and 11.5 mg pantothenate/day; period 3 (21 days), formula diet, providing 2.7 mg pyridoxine and 8.2 mg pantothenate/day. Daily protein intake was 96 g throughout the study. Vitamins in food and urine samples were determined microbiologically and plasma pyridoxal phosphate by a tyrosine apodecarboxylase radioassay method. Compared to the availability of the pure vitamins as 100%, the availability of vitamin B6 ranged from 61 to 81% with a mean of 71% using plasma pyridoxal phosphate data, and ranged from 73 to 92% with a mean of 79% according to urinary vitamin B6 data. Availability of pantothenate ranged from 40 to 61% with a mean of 50%, according to urinary pantothenate data. The average American diet used in our study contained 1.7 and 5.8 mg/day of available vitamin B6 and pantothenate, respectively.