Nitric oxide (NO) is a versatile second messenger. NO is produced by Leydig cells, where NO is a negative regulator of steroidogenesis. In cancer cells, NO is thought to have mutagenic and proliferative effects. We have previously shown that the calcium-sensing receptor (CaR) has promalignant effects in rat H-500 Leydig cancer cells, a model for humoral hypercalcemia of malignancy. Calcium, the major physiological ligand of the CaR, is a recognized intracellular cofactor in the process of NO production by virtue of its positive modulation of neuronal and endothelial nitric oxide synthase (NOS), but importantly, not of inducible (i) NOS activity. iNOS activity is regulated by changes in its expression level. Therefore, we investigated whether CaR activation changes iNOS expression. We found that high extracellular calcium (Ca_o ²⁺) upregulates the level of mRNA for iNOS, whereas no change was seen in neuronal or endothelial NOS, as assessed by microarray and real-time PCR, respectively. The high Ca_o ²⁺-induced iNOS upregulation was also detected by Northern and Western blotting. By quantitative real-time PCR, we showed that calcium maximally upregulates iNOS at 18 h. The effect of calcium was abolished by overexpression of a dominant-negative CaR (R185Q), confirming that the effect of Ca_o ²⁺ was mediated by the CaR. Cells treated with high calcium had higher NO production than those treated with low calcium, as detected with the NO-specific DAF2-AM dye. This was confirmed in single-cell fluorescence determinations using confocal microscopy. In conclusion, high calcium upregulates the levels of iNOS mRNA and protein as well as NO production in H-500 cells, and the effect of Ca_o ²⁺ on iNOS expression is mediated by the CaR.