We examined how previous vitamin D3 intake affects serum 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) after the abrupt start of moderate (10 nmol/day) vitamin D3 doses. Diet calcium and phosphate were normal, but vitamin D3 content differed among groups. In the rats mildly deprived of dietary vitamin D, serum 1,25(OH)2D3 was normal initially, but after the first vitamin D3 dose, serum 1,25(OH)2D3 increased 3-fold, and returned to normal by the second day. In the rats that had been fed normal (2 nmol/day) or elevated (20 nmol/day) vitamin D3, the vitamin D3 doses did not produce the transient rise in 1,25-(OH)2D3. Before the experiment, rats fed diet with 20 nmol vitamin D3/day had lower 1,25(OH)2D3 than the other groups (P<0.05). All groups given vitamin D3 doses for 7 days had lower serum 1,25(OH)2D3 by the end of the week (p<0.05 each). At the end of the study, serum 1,25-(OH)2D3 correlated inversely with serum 25-(OH)D3 (r=-0.608, p<0.01, n=28). There were no differences in weight gain, serum calcium or phosphate among the groups. In conclusion, vitamin D3 doses caused a dramatic, acute rise in serum 1,25(OH)2D3 only in rats fed less than the recommended dietary vitamin D. In contrast, a long-term increase in vitamin D3 intake resulted in lower serum 1,25(OH)2D3, and this was evident in every aspect of this study.