The hormonal form of vitamin D, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], inhibits the proliferation of T lymphocytes and production of growth-promoting factors (including interleukin-2) (IL2) in CTLL2 murine cells. In this study, we investigated the role of monocytes in this hormone-mediated inhibitory effect, by testing the effects of 1,25-(OH)2D3 on the ability of the mitogenic lectin phytohemagglutinin (PHA) to induce T cell activation in either a monocyte-dependent or phorbol myristate acetate (PMA)-driven (monocyte-independent) system. The results indicate that proliferation of T cells and production of growth-promoting factors are inhibited by 1,25-(OH)2D3 only in the monocyte-dependent system. Preincubation of monocytes with 1,25-(OH)2D3 for various periods of time and subsequent removal of the hormone resulted in inhibition of the PHA-driven proliferation of T cells. Preincubation for 2 h resulted in 20% inhibition, while preincubation for 36 h reduced proliferation to 50% of the control value [no 1,25-(OH)2D3 exposure]. These data suggested that monocytes are important participants in 1,25-(OH)2D3-mediated events. Therefore, we tested the effects of the hormone on the production of IL1, a monocyte-derived product thought to be involved in the induction of IL2 release and the subsequent development of the T cell proliferative response. 1,25-(OH)2D3 inhibited the production of both extracellular and cell-associated immunoreactive IL1 alpha and IL1 beta. Indomethacin, a prostaglandin synthetase inhibitor, did not alter the inhibitory properties of 1,25-(OH)2D3, suggesting that prostaglandins are not responsible for the inhibitory phenomenon. We conclude that part of the ability of 1,25-(OH)2D3 to inhibit T cell proliferation may be due to direct effects on monocytes by down-regulating IL-1 production. However, it is unlikely that the immunoregulatory properties of 1,25-(OH)2D3 on T cells are mediated solely through monocytes, and it is possible that the hormone also exerts its influence directly on T cells.