p56(dok-2) acts as a multiple docking protein downstream of receptor or non-receptor tyrosine kinases. However, the role of p56(dok-2) in biological functions of cells is not clear. We found that transcription of the p56(dok-2) gene in macrophages was increased markedly in response to cytokines such as macrophage colony-stimulating factor (M-CSF), granulocyte/macrophage-CSF and interleukin-3 (IL-3). Forced expression of p56(dok-2) inhibited M-CSF-, granulocyte-CSF-, IL-3- and stem cell factor-induced proliferation of myeloid leukemia cells, M-NFS-60. The p56(dok-2)-overexpressing cells showed an impaired induction of c-myc but not of c-jun, junB or c-fos when stimulated with M-CSF. Consistent with these results, the peritoneal cavity of the hairless (hr/hr) strain of mutant mice, whose cells expressed less p56(dok-2) than wild-type mice, contained more macrophages than that of +/hr mice. Moreover, the inhibition of endogenous p56(dok-2) expression in macrophage-like tumor cells, J774A.1, by stable expression of antisense p56(dok-2) mRNA accelerated cell proliferation. The study identifies a novel role for p56(dok-2) as a molecule that negatively regulates signal transduction and cell proliferation mediated by cytokines in a feedback loop.