Urinary excretion of 4-pyridoxic acid (4PA) in 19 men (n = 5) and women (n = 14) was measured to evaluate the validity of determining the 4PA/creatinine ratio in random urine samples as an alternative to total 24-h 4PA excretion in assessing vitamin B6 nutritional status. The relationships among dietary vitamin B6 intake, 4PA excretion, plasma pyridoxal 5'-phosphate levels, and erythrocyte aspartate aminotransferase activity and in vitro stimulation by added plasma pyridoxal 5'-phosphate were examined. The subjects consumed all meals for 3 days in a metabolic unit, and protein intake was kept constant. Plasma pyridoxal 5'-phosphate concentration was positively correlated with vitamin B6 intake of the previous day (r = 0.61, p less than 0.01). There was a positive correlation (r = 0.59, p less than 0.01) between total 4PA and 4PA/creatinine in the 24-h urine samples. No difference (p greater than 0.05) in 4PA/creatinine between the 24-h samples and either morning or afternoon random samples taken the next day was found. These findings support the use of the 4PA/creatinine ratio in random urine samples as an alternative to 24-h urinary 4PA excretion.