Serum ionized calcium (Ca), but not inorganic phosphorus or immunoreactive parathyroid hormone, negatively correlates with renal 25-hydroxyvitamin D- 1α-hydroxylase (1α-hydroxylase) and serum 1,25-dihydroxyvitamin D in intact lactating rats. The present study tested the hypothesis that the presumed stimulation of renal 1α-hydroxylase by hypocalcemia requires the presence of intact parathyroid glands. Lactating and nonlactating rats were surgically parathyroidectomized (PTX) or sham-operated (sham) at 9-10 days of lactation. Later (24 h) the rats were bled, nephrectomized, and killed. In lactating PTX rats, serum ionized Ca decreased to 50% of the level of sham rats, and serum 1,25-dihydroxyvitamin D fell to 37 ± 5.0 pg/ml compared with 82 ± 13.0 pg/ml for sham lactating rats but was still 2.5 times the value for nonlactating PTX rats (15 ± 0.8 pg/ml). In contrast to the still elevated serum 1,25-dihydroxyvitamin D concentration in lactating PTX rats, renal 1α- hydroxylase was suppressed to the same low level as in nonlactating PTX rats, suggesting the existence of extrarenal synthesis of 1,25-dihydroxyvitamin D in lactation. A curvilinear relationship was revealed between serum ionized Ca and renal 1α-hydroxylase in sham lactating and nonlactating rats (r2 = 0.71, P < 0.0001). However, in PTX rats, decreasing ionized Ca did not lead to any increase in 1α-hydroxylase above the low baseline values seen at ionized Ca concentrations between 1.3 and 1.5 mM. We therefore conclude that intact parathyroid glands are required for hypocalcemia to activate renal 1α-hydroxylase in female rats.