Aging is attended by both decreased levels of circulating 1,25-dihydroxy-vitamin D (1,25(OH)2D) and alterations of immune function. We have explored the relationship of these events via the effects of the steroid hormone on macrophage differentiation, using both the human leukemic cell line HL-60, which has the capacity to differentiate along a monocytic or granulocytic pathway, and authentic bone-marrow-derived macrophage precursors. When treated with 1,25(OH)2D, HL-60 cells undergo monocytic differentiation, as documented by the appearance of macrophage-specific membrane antigens and esterase activity. Also, 1,25(OH)2D increases [Ca2+]i in a slow tonic manner, an event that parallels f-Met-Leu-Phe (fMLP) receptor expression. The rise of [Ca2+]i is derived from influx of extracellular Ca2+ and is associated with increased inositol trisphosphate (IP3)-stimulated Ca2+ release from intracellular stores. On the other hand, while prevention of the 1,25(OH)2D-generated increase in [Ca2+]i leads to reduced superoxide generation, it does not block monocytic differentiation. 1,25(OH)2D also targets to authentic bone-marrow-derived macrophage precursors at all stages of differentiation. In CSF-1-dependent cells, the steroid produces doubling of expression of the mannose receptor, a macrophage-specific membrane protein, which is also expressed by differentiated osteoclasts. The macrophage-maturing effect of 1,25(OH)2D was further explored by analyzing its effect on fMLP signal transduction in HL-60 cells. While virgin HL-60 cells are unresponsive to fMLP, cells incubated for 24 h with 1,25(OH)2D respond to fMLP stimulation with a 60% increase in [Ca2+]i, and possess greater IP3-sensitive calcium stores than virgin cells.(ABSTRACT TRUNCATED AT 250 WORDS)