GM-CSF is a colony stimulating factor which regulates growth and differentiation of hematopoietic progenitors. Also, it stimulates increases in c-jun expression and activates AP-1 enhancer activity. However, it is not clearly known how its signal is transduced. In this study, the effect of GM-CSF on tyrosyl phosphorylation was examined in Human U937 and HL-60 Leukemic cells by Western Blotting using anti-phosphotyrosine monoclonal antibody and autoradiography. Evidence is presented that GM-CSF stimulates a rapid phosphorylation on tyrosines of several proteins of relative Mr. 150, 110, 95, 55, 48, and 15 kDa within 60 seconds. The effect of GM-CSF was blocked by the tyrosine kinase inhibitor ST638 but not by H-7. Both G-CSF and M-CSF were also effective in stimulating tyrosine phosphorylation. These results strongly support a role for tyrosine kinases in regulation of cellular events by GM-CSF in monoblasts. © 1993 Academic Press, Inc.