The calcium-regulating hormone, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), is also recognized as an immunomodulator. In vitro addition of the hormone to bone marrow-derived macrophages (BMDMs) results in a decreased proliferation and an increased differentiation. In the present work we compare the in vitro differentiation of BMDMs derived from vitamin D-depleted and -repleted mice (-D and +D BMDMs, respectively). -D BMDMs proliferate in vitro slower than +D BMDMs. Addition of the hormone to BMDM cultures inhibited the rate of their proliferation, which was more pronounced in low-density cultures. The ability of mononuclear phagocytes to produce reactive oxygen metabolites is an important element in the microbicidal functions of these cells. We found that -D BMDMs produce less H2O2 than +D BMDMs, which was corrected by the in vitro addition of 1,25(OH)2D3. Analyses of various macrophage-specific surface antigens revealed a reduction in their expression on -D BMDMs. In vitro addition of 1,25(OH)2D3 to BMDM cultures increased the expression of these antigens. The activity of the lysosomal enzyme acid phosphatase was similarly affected by vitamin D deficiency and by the in vitro addition of the hormone. Thus, vitamin D deficiency is associated with impaired maturation of BMDMs suggesting that the hormone is a natural modulator of macrophage maturation.