To determine the renal tubular site and mechanism of the anticalciuric effects of trichloromethiazide (TCM) and amiloride, we studied the effects of these diuretics on net Ca2+ absorption (JCa) in isolated rabbit connecting tubules (CNT) and cortical collecting ducts (CCD). TCM (100 microM) in the lumen increased JCa in the CNT without affecting lumen-negative transepithelial voltage (VT). This effect was dependent on the amount of parathyroid hormone (PTH, 0.1 or 1 nM) in the bath. TCM had no detectable effect on JCa in the absence of PTH. Addition of 100 microM ouabain to the bath decreased PTH-stimulated JCa and abolished the TCM-stimulated JCa. Elimination of Na+ from the lumen increased PTH-stimulated JCa. Under this condition the ability of TCM to increase JCa was abolished, suggesting that the inhibition of Na+ entry from the lumen with TCM may account for the increase in PTH-stimulated JCa. Elimination of Na+ from the bath in the presence of PTH decreased JCa and abolished the stimulatory effect of TCM on JCa in the presence of PTH. Changes in VT caused by amiloride may not account for the increase in JCa, because JCa was not changed when VT was deflected to more positive direction by increasing bath K+ from 5 to 15 mM. Neither TCM nor amiloride affected JCa in the CCD. From these observations, we speculate that the inhibition of Na+ entry across the apical cell membrane by either thiazides or amiloride may stimulate PTH-induced JCa. The intact Na(+)-Ca2+ antiporter in the basolateral cell membrane is essential for the anticalciuric effect of thiazides and amiloride.