Both calcium and 1,25(OH)₂D promote the differentiation of keratinocytes in vitro. The autocrine or paracrine production of 1,25(OH)₂D by keratinocytes combined with the critical role of the epidermal calcium gradient in regulating keratinocyte differentiation in vivo suggest the physiologic importance of this interaction. The interactions occur at a number of levels. Calcium and 1,25(OH)₂D synergistically induce involucrin, a protein critical for cornified envelope formation. The involucrin promoter contains an AP-1 site essential for calcium and 1,25(OH)₂D induction and an adjacent VDRE essential for 1,25(OH)₂D but not calcium induction. Calcium regulates coactivator complexes that bind to the Vitamin D receptor (VDR). Nuclear extracts from cells grown in low calcium contain an abundance of DRIP₂₀₅, whereas calcium induced differentiation leads to reduced DRIP₂₀₅ and increased SRC 3 which replaces DRIP in its binding to the VDR. In vivo models support the importance of 1,25(OH)₂D–calcium interactions in epidermal differentiation. The epidermis of 1αOHase null mice fails to form a normal calcium gradient, has reduced expression of proteins critical for barrier function, and shows little recovery of the permeability barrier when disrupted. Thus in vivo and in vitro, calcium and 1,25(OH)₂D interact at multiple levels to regulate epidermal differentiation.