We investigated the influence of interferon-alpha (IFN-alpha) on the synthesis of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) by monocytes and activated T helper cells. IFN-alpha inhibited the production of GM-CSF in unstimulated and lipopolysaccharide (LPS)-activated monocytes to the same extent as was observed in the presence of IL-4. In highly purified CD4+ T cells, which were activated by incubation with immobilized anti-CD3 antibody and anti-CD28, IFN-alpha reduced production of GM-CSF to 47%. In contrast, GM-CSF production in activated T cells was unaffected by exogenously added IL-4. The production of IL-3 by T helper cells was significantly inhibited by IFN-alpha as well. IL-3 production by CD3/CD28-stimulated T helper cells was exclusively enhanced by IL-4. The exogenous addition of IL-4 led to a highly significant increase of IL-3 levels in T cell supernatants to 231% of control cultures (range 137%-605%), whereas other T cell-derived cytokines, such as IFN-gamma and IL-10, failed to influence IL-3 release. The differential role of IL-4 in IL-3 production was confirmed by the addition of anti-IL-4 antibodies to CD3/CD28-stimulated T cells. Neutralizing anti-IL-4 antibody caused a drastic reduction of IL-3 synthesis by activated T cells, whereas GM-CSF production was independent of neutralization of endogenous IL-4. These experiments define IFN-alpha as an inhibitory substance for the production of hematopoietic growth factors by activated immune cells. The influence of IL-4 on cytokine synthesis appears to be cell type specific, thus revealing a differential stimulatory effect on IL-3 production.