The immunomodulatory effects of vitamin D have been described following chronic oral administration to mice or supplementation of cell cultures with 1,25-dihydroxyvitamin D-3 (1,25(OH)(2)D-3)1 the active form of vitamin D. In this study, topically applied 1,25(OH)(2)D-3, enhanced the suppressive capacity of CD4(+)CD25(+) cells from the draining lymph nodes. The effects of topical 1-25(OH)(2)D-3 were compared with those of UVB irradiation, which is the environmental factor required for 1,25(OH)(2)D-3 production in skin. CD4(+) cells from the skin-draining lymph nodes (SDLN) of either 1,25(OH)(2)D-3-treated or UVB-irradiated mice had reduced capacity to proliferate to Ags presented in vitro, and could suppress Ag-specific immune responses upon adoptive transfer into naive mice. This regulation was lost upon removal of CD4(+)CD25(+) cells. Furthermore, purified CD4(+)CD25(+) cells from the SDLN of 1,25(OH)(2)D-3-treated or UVB-irradiated mice compared with equal numbers of CD4(+)CD25(+) cells from control mice had increased capacity to suppress immune responses in both in vitro and in vivo assay systems. Following the sensitization of recipient mice with OVA, the proportion of CD4(+)Foxp3(+) cells of donor origin significantly increased in recipients of CD4(+)CD25(+) cells from the SDLN of 1,25(OH)2D3-treated mice, indicating that these regulatory T cells can expand in vivo with antigenic stimulation. These studies suggest that 1,25(OH)(2)D-3 may be an important mediator by which UVB-irradiation exerts some of its immunomodulatory effects.