Inorganic phosphate (Pi) is absorbed by proximal tubules through a cellular pathway that is inhibited by parathyroid hormone (PTH). The calcium-sensing receptor (CaSR) is expressed on apical membranes of proximal tubules. In the present studies, we determined the effect of luminal and/or basolateral PTH on phosphate absorption and tested the hypothesis that CaSR activation blocks PTH-inhibitable phosphate absorption. Single proximal S3 tubules were dissected from the kidneys of mice and studied by the Burg technique. Tubules were bathed with DMEM culture media supplemented with 6% BSA and perfused with an ultrafiltrate prepared from the bathing solution. 33P and FITC-inulin were added to the luminal perfusate to measure phosphate absorption (JPi) and fluid absorption (Jv), respectively. JPi averaged 2.9 pmol.min-1.mm-1 under control conditions and decreased by 20% upon addition of serosal PTH. PTH had no effect on Jv. Inclusion of PTH in the luminal perfusate reduced JPi to 2.1 pmol. min-1. mm-1. Combined addition of PTH to perfusate and bathing solutions reduced JPi to 1.5 pmol. min-1. mm-1 without affecting Jv. Indirect immunofluorescence studies revealed abundant PTH receptor (PTH1R) expression on brush-border membranes, with lower amounts on basolateral membranes. CaSRs were localized primarily, but not exclusively, to brush-border membranes. CaSR activation with luminal Gd3+ abolished the inhibitory action of PTH on JPi. Addition of Gd3+ to the serosal bathing solution had no effect on PTH-sensitive JPi. Gd3+ i.e., PTH-independent JPi. Gd3+ did not affect basal, had no effect on Jv when added to lumen or bath. Dopamine-inhibitable JPi was not affected by Gd3+. Experiments with proximal-like opossum kidney cells showed that elevated extracellular Ca2+ or NPS R467, a type II calcimimetic, inhibited PTH action on Pi uptake. In conclusion, PTH1Rs are expressed on apical and basolateral membranes of mouse proximal tubules. Stimulating apical or basolateral PTH1R inhibits phosphate absorption. CaSR activation specifically regulates PTH-suppressible phosphate absorption.