To better understand the extrarenal production of active vitamin D metabolites by cells of the monocyte/macrophage lineage, we investigated the 25-hydroxyvitamin D (25OHD)-1-hydroxylation reaction in the v-myc-transformed chick myelomonocytic cell line HD-11; the 1-hydroxylation reaction in this cell line has a high affinity for 25-hydroxylated vitamin D substrates, is localized to mitochondria, and is associated with cytochrome P450 activity. In this study we demonstrated that the HD-11 cell 1-hydroxylation reaction in vitro is not affected by the majority of extracellular regulatory factors that modulate expression of the renal 25OHD-1-hydroxylase in vivo. A 50% increase in extracellular calcium and phosphate concentrations, physiological inhibitory events for renal 1,25-dihydroxyvitamin D [1,25-(OH)2D] synthesis, did not decrease basal expression of the HD-11 cell 1-hydroxylation reaction, nor did a 50% decrease in extracellular calcium and phosphate concentrations, stimulatory signals for the 1-hydroxylase in vivo, increase 1,25-(OH)2D3 synthesis in vitro. Receptor-saturating concentrations of PTH and PTH- related peptide were similarly without effect. In contrast, the HD-11 1- hydroxylation reaction was significantly stimulated in a dose-dependent fashion by the macrophage stimulatory agents lipopolysaccharide [P < 0.001 at a maximum effective concentration (EC100) of 25 μg/ml] and interferon-γ (P < 0.001 at EC100 of 1000 IU/ml) and by insulin-like growth factor-I (P < 0.01 at EC100 of 15 nM) with the rank order of stimulation being interferon-γ > lipopolysaccharide > insulin-like growth factor-I. Dexamethasone (≥10 nM) and the cytochrome P450 inhibitors (EC100, 20 μM), ketoconazole, clotrimazole, and menadione, all significantly inhibited the HD-11 cell 1-hydroxylation reaction. The napthoquinone menadione, which blocks electron transfer to the P450-associated enzyme, was the most effective inhibitor of the reaction in both intact cells (3 ± 1% of basal expression; P ≤ 0.002) and after reconstitution of HD-11 cell mitochondrial extracts with a ferredoxin, reductase, O2, and NADPH (5 ± 1% of basal; P ≤ 0.02). We have also shown that 1,25-(OH)2D3 produced from substrate 25OHD3 appears to exert an endogenous (intracrine) inhibitory effect on HD-11 cell growth; incubation of HD-11 cells with a concentration of ketoconazole (10 μM) known to reduce 1,25-(OH)2D3 production by roughly 50% restored 50% of the growth deficit induced by 1,25-(OH)2D3 (EC100, 100 nM). These results suggest that expression of the macrophage 25OHD3-1-hydroxylation reaction is 1) dependent upon functional integrity of the cell's compliment of cytochrome P450, 2) regulated most effectively by extracellular mediators of inflammatory cell activity, and 3) capable of exerting an intracrine growth inhibitory effect.