Extracellular calcium (Cao) stimulates the differentiation of keratinocytes; 1,25 dihydroxyvitamin D3 1,25(OH)2D3) does likewise. Since 1,25(OH)2D3 regulates calcium flux in other cells, we hypothesized that 1,25(OH)2D)3 acted through and promoted the effects of calcium on keratinocyte differentiation. To test this hypothesis, we evaluated the effects of calcium and 1,25(OH)2D3 alone and in combination on the mRNA and protein levels of involucrin and transglutaminase in neonatal human keratinocytes. Cao alone increased these mRNA levels in a dose-dependent fashion (0.03 to 1.2 mM) over a 24-h period. This increase in mRNA levels was associated with a stimulation by calcium of involucrin and transglutaminase gene transcription. However, by 72 h, the mRNA levels of involucrin and transglutaminase decreased. At 0.03 mM Cao, 1,25(OH)2D3 showed a dose-dependent stimulation of involucrin and transglutaminase mRNA for up to 48 h and potentiated the initial (4-h) stimulation by Cao of involucrin and transglutaminase mRNA. As for calcium alone, this increase in mRNA was associated with an increase in transcription of the involucrin and transglutaminase genes. However, by 24 h of exposure to both calcium and 1,25(OH)2D3, a dose-dependent fall in mRNA levels was seen. The mRNA levels of involucrin and transglutaminase were stable for 24 h when neonatal human keratinocytes were grown in serum-free keratinocyte growth medium containing 0.03 or 1.2 mM Cao alone. However, the mRNAs of both genes underwent rapid degradation when neonatal human keratinocytes were treated with 1,25(OH)2D3, especially in high Cao. 1,25(OH)2D3 and Cao increased the protein levels of involucrin and transglutaminase activity in a synergistic fashion throughout the 48-h time course. These data support the hypothesis that 1,25(OH)2D3 promotes calcium-induced differentiation at the level of both gene expression and mRNA stability.