Humoral hypercalcemia of malignancy (HHM) occurs when secretion of parathyroid hormone-related peptide (PTHrP) by cancer cells causes hypercalcemia in the absence of skeletal metastases. High extracellular calcium (Ca-o(2+)) increases secretion of PTH-like bioactivity by rat H-500 leydig cells, a transplantable model of HHM, an action potentially mediated by the Ca-o(2+)-sensing receptor (CaR). In this study we investigated whether H-500 cells express the CaR and, if so, whether CaR agonists modulate PTHrP secretion. Northern blot analysis and RT-PCR revealed bona fide CaR transcript(s), and immunocytochemistry and Western analysis with a specific anti-CaR antiserum demonstrated CaR protein expression in H-500 cells. Furthermore, high Ca-o(2+) and neomycin stimulated PTHrP secretion dose-dependently with maximal 2.7- and 3.3-fold increases at 5 mM Ca-o(2+) and 300 mu M neomycin, respectively. Thus in HHM caused by H-500 cells, the CaR could participate in a vicious cycle whereby PTHrP-induced increases in Ca-o(2+) further stimulate PTHrP release and exacerbate hypercalcemia.