The hormonal form of vitamin D₃, 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃], at picomolar concentrations, inhibited the growth-promoting lymphokine interleukin-2, which is produced by human T lymphocytes activated in vitro by the mitogen phytohemagglutinin. Other metabolites of vitamin D₃ were less effective than 1,25(OH)₂D₃ in suppressing interleukin-2; their order of potency corresponded to their respective affinity for the 1,25(OH)₂D₃ receptor, suggesting that the effect on interleukin-2 was mediated by this specific receptor. The proliferation of mitogen-activated lymphocytes was also inhibited by 1,25(OH)₂D₃. This effect of the hormone became more pronounced at later stages of the culture. These findings demonstrate that 1,25(OH)₂D₃ is an immunoregulatory hormone.