The physiological role of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] in inhibition of its own formation remains obscure. This study utilizes a kidney slice system to study the effect of physiological concentrations of 1,25(OH)2D3 on 25-hydroxyvitamin D3 1-hydroxylase (1-hydroxylase) formation in vitamin D-replete rats fed a normal-phosphate (NP) or low-phosphate (LP) diet. In vitro, 1-hydroxylase activity was assessed by measuring 1,25(OH)2D3 accumulation at 30 or 60 min after addition of 25-hydroxyvitamin D3 substrate. Degradation of 1,25(OH)2D3 was also assessed over 60 min. Rats fed the LP diet had a threefold increase in 1-hydroxylase activity but the same rate of degradation of 1,25(OH)2D3 as those fed the NP diet. The addition of 50 pM 1,25(OH)2D3 caused a proportional inhibition of 1-hydroxylase in NP and LP rats when added before or 10 min after addition of substrate but not at later time points; 150 pM 1,25(OH)2D3 completely inhibited 1-hydroxylase in the NP but not the LP rats. This inhibitory effect was not reversed by actinomycin D or cycloheximide. These results indicate that physiological concentrations of 1,25(OH)2D3 directly and rapidly modulate 1-hydroxylase activity via a nongenomic mechanism in both LP and NP diet rats.