Bioactive PTH was measured in Wistar rats under a variety of experimental conditions. The mean activity in normal rat sera was 0.17 +/- 0.12 ng/ml (expressed in terms of bovine PTH 1-34). Sera from animals reared on a vitamin D deficient diet showed a mean value of 0.46 +/- 0.24 ng/ml (P less than 0.01), whereas sera from animals with 1,25-dihydroxyvitamin D (1,25(OH)2D) deficiency had a mean activity of 0.62 +/- 0.23 ng/ml (P less than 0.01). Dietary calcium deficiency also resulted in high serum PTH levels (0.71 +/- 0.34 ng/ml, P less than 0.01) in spite of marked elevations of serum 1,25(OH)2D concentrations in these animals. A significant negative correlation was noted between serum calcium and bioactive PTH. Calcium infusions into hypocalcemic, vitamin D-deficient rats caused a fall in serum bioactive PTH concentrations to a mean of 13% of control values within 10 min. Intraperitoneal administration of 1,25(OH)2D3 to hypocalcemic, 1,25(OH)2D-deficient rats did not suppress serum bioactive PTH concentrations after 30 or 60 min even though serum 1,25(OH)2D concentrations were greater than 900 pmol/liter in each animal at these time points. To our knowledge, this is the first study using PTH bioassays for physiological experiments in rats.