The steroid hormone 1,25 dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) regulates cell proliferation and differentiation. Intracellular calcium (Cai) concentrations play a crucial role in these events. From our previous studies, we have demonstrated a calcium receptor (CaR) in keratinocytes which appears to regulate the initial release of Cai from intracellular stores in response to extracellular calcium (Cao) and so is likely to participate in the differentiation process. In this study, we determined whether the ability of 1,25(OH) 2 D 3 to enhance Ca ฯฉฯฉ -induced differentiation was mediated at least in part through changes in the CaR. Keratinocytes were grown in keratinocyte growth medium (KGM) with 0.03 mM, 0.1 mM, or 1.2 mM Ca and treated with 10 -8 M 1,25(OH) 2 D 3 till harvest after 5, 7, 14, and 21 days. CaR mRNA levels were quantitated by polymerase chain reaction. The results were compared to the ability of 1,25(OH) 2 D 3 to enhance calcium-stimulated increases in Cai. In cells grown in 0.03 mM Ca, the CaR mRNA levels decreased with time. 1,25(OH) 2 D 3 stimulated the levels at 5 days and prevented the falloff over the subsequent 16 days. On the other hand, in cells grown in 0.1 or 1.2 mM Ca, the message levels remained high, and 1,25(OH) 2 D 3 had no further effect. To study the functional relationship, we harvested cells after 5 and 7 days in culture following a 24 h treatment with 1,25(OH) 2 D 3 or vehicle to measure the Cai response to 2 mM Cao. The preconfluent cells grown in 0.03 mM Ca showed a nearly twofold increase in the Cai response to Cao when pretreated with 1,25(OH) 2 D 3 , whereas the confluent cells and those grown in 1.2 mM Ca showed no enhancement by 1,25(OH) 2 D 3 . Studies with 45 Ca influx into keratinocytes revealed that 1,25(OH) 2 D 3 enhanced the influx in preconfluent and confluent cells when grown in KGM containing 0.03 mM Ca but not in cells grown in 1.2 mM calcium. We conclude that 1,25(OH) 2 D 3 maintains the CaR mRNA levels in cells grown in 0.03 mM Ca, thus maintaining their responsiveness to Cao and so ensuring their ability to differentiate in response to the calcium signal.