The effects of p40x, a product of an human T cell leukemia virus type I, on the activation of lymphokine genes were examined. The mouse GM-CSF and IL-3 genes were activated by cotransfection with a pX containing plasmid both in Jurkat and CV1 cells. Mouse GM-CSF gene was also activated by phytohaemagglutinin A (PHA)/phorbol myristate acetate (PMA) or PMA/calcium ionophore A23187 stimulation. The 5'-flanking region of the mouse GM-CSF gene which is required for activation by pX or mitogen was mapped within 226 bp upstream from the transcription initiation site. Action of pX was not restricted to T cells. pX activated exogenously added GM-CSF, IL-2, IL-3 and IL-4 genes in fibroblasts. Activation of the GM-CSF gene in fibroblasts appears to require the same regulatory region as in T cells. Similar results were obtained using bovine papilloma virus encoded E2 protein. We propose that pX or E2 protein, both in T cells and fibroblasts, activates cellular component(s) in the signal transduction pathway which results in the activation of lymphokine genes in the absence of extracellular stimuli.