Previous studies in chondrogenic RCJ3.1C5.18 (C5.18) cells showed that growth of these cells at high extracellular Ca²⁺ concentrations ([Ca²⁺]_o) reduced the expression of markers of early chondrocyte differentiation. These studies addressed whether raising [Ca²⁺]_o accelerates C5.18 cell differentiation and whether Ca²⁺ receptors (CaRs) are involved in coupling changes in [Ca²⁺]_o to cellular responses. We found that high [Ca²⁺]_o increased expression of osteopontin (OP), osteonectin, and osteocalcin, all markers of terminal differentiation, in C5.18 cells and increased the production of matrix mineral. Overexpression of wild-type CaR cDNA in C5.18 cells suppressed proteoglycan synthesis and aggrecan RNA, two early differentiation markers, and increased OP expression. The sensitivity of these parameters to changes in [Ca²⁺]_o was significantly increased, as indicated by left-shifted dose-responses. In contrast, stable expression of a signaling-defective CaR mutant (Phe707Trp CaR) in C5.18 cells, presumably through dominant-negative inhibition of endogenous CaRs, blocked the suppression of aggrecan RNA levels and proteoglycan accumulation and the enhancement of OP expression by high [Ca²⁺]_o. These data support a role for CaRs in mediating high [Ca²⁺]_o-induced differentiation of C5.18 cells.