The availability of Ca²⁺ in the extracellular fluid plays an important role in regulating cartilage and bone formation. We hypothesized that chondrocytes detect changes in the extracellular[ Ca²⁺] ([Ca²⁺]_o) and modify their function. The effects of changing[ Ca²⁺]_o on the expression of matrix proteins were quantified by staining of cartilage nodules with alcian green and assessing RNA levels of cartilage-specific genes in chondrogenic RCJ3.1C5.18 (C5.18) cells. Alcian green staining in these cells decreased with increasing [Ca²⁺]_o in a dose-dependent and reversible manner (ID₅₀, ∼2 mm Ca²⁺). RNA levels for aggrecan and type II collagen decreased with increasing [Ca²⁺]_o (ID₅₀, ∼2.0 and 4.1 mm Ca²⁺, respectively). RNA levels for type X collagen and alkaline phosphatase were also reduced by high [Ca²⁺]_o with ID₅₀ values of approximately 2.9 and 1.6 mm Ca²⁺, respectively. These responses were rapid, in that increasing [Ca²⁺]_o from 1.0 to more than 6 mm suppressed aggrecan RNA levels by about 50%, and lowering [Ca²⁺]_o from 2.9 to 1.0 mm increased aggrecan RNA levels by about 300% within 4 h. As Ca²⁺ receptors (CaRs) mediate extracellular Ca²⁺ sensing in parathyroid and kidney, we assessed the expression of CaRs in these cells. C5.18 cells stained positively for CaR protein with an anti-CaR antiserum and for CaR RNA by in situ hybridization. An approximately 150-kDa protein was detected by immunoblotting with anti-CaR antiserum. CaR antisense oligonucleotides suppressed the expression of CaR protein and enhanced RNA levels of aggrecan in C5.18 cells. These data support the idea that CaRs are expressed in this cell system and may be involved in regulating chondrogenic gene expression.