The immunomodulatory hormone 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) has been shown to suppress T-cell proliferation and interleukin-2 synthesis as well as B-cell immunoglobulin synthesis, while stimulating many macrophage functions. We have previously shown increased synthesis of interleukin-1 beta in lipopolysaccharide (LPS)-stimulated U937 cells after pretreatment with 10 nmol/L 1,25-(OH)2D3. We now show that 1,25-(OH)2D3 also primes the increase in U937 cell tumor necrosis factor (TNF)-alpha-accumulated mRNA after activation with LPS; 50% effective concentration (EC50) for the LPS-induced expression of TNF-alpha mRNA was decreased by two orders of magnitude after incubation with 10 nmol/L 1,25-(OH)2D3. Pretreatment of U937 cells with 10 nmol/L 1,25-(OH)2D3 also increased subsequent LPS-induced TNF-alpha mRNA expression by twofold and cell-associated TNF protein levels by more than ninefold. This potentiation was steroid-specific for 1,25-(OH)2D3 because dexamethasone inhibited TNF-alpha mRNA. The potentiation required prior exposure to 1,25-(OH)2D3 for more than 6 hours and was clearly seen after 12 hours. The finding that the sensitivity of the U937 cell monokine response to LPS was dramatically increased by 1,25-(OH)2D3 and the delayed effect on the LPS-stimulated TNF-alpha gene transcript levels indicated that 1,25-(OH)2D3 may be altering the expression of a protein(s) in the U937 cell LPS-signal transduction pathway. In fact, 1,25-(OH)2D3 induced expression of the mRNA for CD14, the high affinity, cell-surface glycoprotein receptor for LPS, which could account for the enhancement of LPS-stimulated monokine gene expression by 1,25-(OH)2D3. Thus, local monokine gene expression may be regulated by both the amount and the temporal entry of the vitamin D hormone and activator(s) into the inflammatory microenvironment.