We recently reported that in healthy men, changes in the production rate (PR) of 1,25-dihydroxyvitamin D [1,25-(OH)2D] accounted for the 80% increase and the 30% decrease in its serum concentration that was induced by restriction and supplementation, respectively, of dietary phosphorus. These changes in PR and serum concentration of 1,25-(OH)2D could be mediated by changes in serum concentrations of phosphorus that occur after the morning fasting period. To examine this hypothesis, we measured serum concentrations of phosphorus in blood drawn at hourly intervals for 24 h in six healthy men in whom dietary phosphorus was initially maintained at 1,500 mg/70 kg body weight per day for 9 d, then restricted to 500 mg/d (coupled with orally administered aluminum hydroxide) for 10 d, and then supplemented to 3,000 mg/d for 10 d. When dietary phosphorus was normal, the serum concentration of phosphorus exhibited the normal circadian rhythm: a rapid decrease in early morning to a nadir at 1100, followed by an increase to plateau at 1600 h and a further increase to an acrophase (peak) at 0030 h. The variation in serum levels of phosphorus can be described as the sum of sinusoidal functions with periodicities of 24 and 12 h. Phosphorus restriction for 10 d induced a 40% reduction in the 24-h mean serum level of phosphorus, abolished the early afternoon rise in its serum level (i.e., the 12-h periodic component of the time series), and delayed the acrophase by 3 h to 0330 h. Phosphorus supplementation for 10 d induced a 14% increase in the 24-h mean serum level of phosphorus but no significant change in its morning fasting level, exaggerated the early afternoon rise in serum phosphorus, and advanced the acrophase by 9 h to 1530 h. The changes in the PR of 1,25-(OH)2D induced by restriction and supplementation of dietary phosphorus varied inversely and significantly with those induced in the 24-h mean serum level of phosphorus (R = -0.88, P less than 0.001). These data demonstrate that in healthy men, dietary phosphorus is an important determinant of the serum concentration of phosphorus throughout most of the day. The data suggest that diet-induced changes in serum levels of phosphorus mediate the changes in PR and serum concentration of 1,25(OH)2D.