We have studied the effect of 10(-8) M 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] on insulin binding, insulin receptor gene expression, and isotype mRNA pattern in U-937 human promonocytic cells. Binding assays indicated that 1,25-(OH)2D3 increased total receptor number without altering receptor affinity, while the dissociation rate of the hormone bound to its receptor remained unchanged. RNA blot assays indicated that 1,25-(OH)2D3 increased the levels of the two major insulin receptor mRNAs (11 and 8.5 kb) present in these cells. Experiments with the RNA synthesis inhibitor actinomycin D suggested that 1,25-(OH)2D3 did not alter the stability of total insulin receptor mRNA. Experiments with the protein synthesis inhibitor cycloheximide indicated that the increase in the amounts of this mRNA occurs as a direct response to the action of the hormone. Finally, polymerase chain reaction assays revealed that the insulin receptor mRNA isotype lacking the exon 11 (A isotype) was the only one present in both untreated and 1,25-(OH)2D3-treated U-937 cells. This indicates that 1,25-(OH)2D3 does not alter the splicing of the primary insulin receptor transcript in this cell line.