C57Bl/6 female mice fed a Vitamin D (VIT-D)-deficient diet had serum levels of 25-hydroxyvitamin D decreasing with the time of diet exposure (3 and 8 weeks). Cytokine production (IL-6, TNF and IL-1) by peritoneal macrophages cultured in vitro with a standard stimulus, LPS, evaluated in the supernatants as biological activity, was significantly reduced in VIT-D-deficient animals. The defect in monokine production was partial and was evident at suboptimal LPS concentrations and incubation times. I-A antigen expression, induced in macrophages by in vitro exposure to IFN-gamma, was not modified in VIT-D-deficient mice, but IFN-gamma-inducible macrophage cytotoxicity to tumour target cells was significantly decreased in VIT-D-deficient animals. Moreover, basal and Poly I:C-induced NK activity was not modified by VIT-D deficiency. Thus, macrophage functions, such as cytokine production and tumour cytotoxicity induction, are down-modulated in vitro by VIT-D deprivation. To give more support to the relevance of VIT-D availability for cytokine production, TNF and IL-6 have been evaluated in the sera of control and VIT-D-deficient mice given LPS as a model stimulus. Serum peak levels of both cytokines were at least halved in VIT-D-deprived mice. Thus, VIT-D deficiency may represent a model of partial defect of monokine production.