Proximal convoluted (S2) and straight (S3) renal tubule segments were studied to determine the effect of Ca on lumen-to-bath phosphate flux (JlbPO4). Increasing bath and perfusate Ca from 1.8 to 3.6 mM enhanced JlbPO4 from 3.3 +/- 0.7 to 6.6 +/- 0.6 pmol/mm per min in S2 segments (P less than 0.001) but had no effect in S3 segments. Decreasing bath and perfusate Ca from 1.8 to 0.2 mM reduced JlbPO4 from 3.7 +/- 0.6 to 2.2 +/- 0.6 in S2 segments. These effects were unrelated to changes in fluid absorption and transepithelial potential difference. Increasing cytosolic Ca with a Ca ionophore, inhibiting the Ca-calmodulin complex with trifluoperazine, or applying the Ca channel blocker nifedipine had no effect on JlBPO4 in S2 segments. Increasing only bath Ca from 1.8 to 3.6 mM did not significantly affect JlbPO4. However, increasing only perfusate Ca enhanced JlbPO4 from 3.4 +/- 0.7 to 6.1 +/- 0.7 pmol/mm per min (P less than 0.005). Inhibition of hydrogen ion secretion, by using a low bicarbonate, low pH perfusate, both depressed base-line JlbPO4 and abolished the stimulatory effect of raising perfusate Ca. Net phosphate efflux (JnetPO4) also increased after ambient calcium levels were raised, ruling out a significant increase in PO4 backflux. When net sodium transport was abolished by reducing the bath temperature to 24 degrees C, JnetPO4 at normal ambient calcium was reduced and increasing ambient calcium failed to increase it, ruling out a simple physicochemical reaction wherein phosphate precipitates out of solution with calcium. The present studies provide direct evidence for a stimulatory effect of Ca on sodium-dependent PO4 absorption in the proximal convoluted tubule, exerted at the luminal membrane. It is postulated that Ca modulates the affinity of the PO4 transporter for the anion.