Growth factor independence of hematopoietic cells can be induced by ectopic expression of a variety of oncogenes encoding receptor or cytoplasmic tyrosine kinases. To examine whether the activation of tyrosine kinases occurs in factor-independent mutants in vivo, the tyrosine-phosphorylated proteins from 14 factor-independent mutants of a GM-CSF-dependent cell line (TF-1) were analysed. These mutants did not secrete any growth-stimulating activity for TF-1 cells, suggesting that activation of intracellular signaling rather than an autocrine stimulation by secreted growth factors is responsible for their factor-independent growth. In 11 out of 14 GM-CSF-independent mutants analysed, a constitutively tyrosine-phosphorylated protein of 60 kDa was detected, which was subsequently identified as p60(c-Src). The kinase activity of p60(c-Src) was increased up to 12-fold in these mutants, which was at least in part due to overexpression of the c-src gene on the RNA and protein level. The Src substrate Sam68 showed an increased phosphorylation in mutants with high Src activity, suggesting that p60(c-Src) triggers downstream signaling in these cells. Treatment of the factor-independent mutants with the Src kinase inhibitor PP2 resulted in a reduced proliferation, demonstrating that Src kinases are essential for these cells for maximal proliferation. Further analysis of factor-independent mutants with low or undetectable Src activity revealed a constitutive phosphorylation of the common beta chain of the GM-CSF receptor and STAT5. Our data indicate an increase in the expression and total activity of endogenous p60(c-Src) in several GM-CSF-independent TF-1 mutants, further underlining the role of Src in the process of autonomous growth of hematopoietic cells.