Elevated levels of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) are found in late pregnancy but the factors responsible for this are not known. To determine if the maternal-fetal calcium flux or the presence of a previously described extrarenal 25-hydroxycholecalciferol-1-hydroxylase (25(OH)-D3-1-hydroxylase) play a role, serum calcium and 1,25(OH)2D3 were measured in pregnant, nonpregnant, and decidua-bearing pseudopregnant rats. Serum calcium was 8.74 +/- 0.26 mg/dl (mean +/- SEM) in nonpregnant rats. In pregnant rats, serum calcium was not significantly different from nonpregnant controls on day 12 and only slightly higher on day 15. Pseudopregnant rats were significantly hypercalcemic on days 12 (11.93 +/- 0.19 mg/dl) and 15 (11.45 +/- 0.23 mg/dl) compared with nonpregnant rats (P less than 0.001). In nonpregnant controls the serum level of 1,25(OH)2D3 was 44.6 +/- 6.3 pg/ml. Levels in pregnant rats were not significantly different on days 12 or 15 but tended to be higher by day 15 (75.2 +/- 19.7 pg/ml). Pseudopregnant rats had levels of 72.6 +/- 13.5 pg/ml on day 12 and 102.8 +/- 10.9 pg/ml on day 15, the latter of which was significantly higher than nonpregnant values (P less than 0.05). 25(OH)D3-1-hydroxylase activity was determined in whole tissue homogenates of placenta and decidua. Placenta from pregnant rats and decidua from pregnant and pseudopregnant rats both formed putative 1,25(OH)2D3 in short-term incubation with 25(OH)D3 as identified by comigration with authentic 1,25(OH)2D3 on high pressure liquid chromatography (HPLC).(ABSTRACT TRUNCATED AT 250 WORDS)