ABSTRACT Most of the nine members of the Src family of tyrosine kinases are restricted in their expression, often to cells of the haematopoietic lineage, while some, particularly Src, Fyn and Yes, are more ubiquitously expressed. We have been studying the functions of Src, Fyn and Yes in fibroblasts. We have shown that stimulation of quiescent fibroblasts with platelet-derived growth factor (I’DGF) causes Src, Fyn and Yes to become activated, and to associate transiently with the I’DGF receptor. To address the role of Src, Fyn and Yes in the response to I’DGF, we have used a dominant negative approach, in which cells were engineered to express catalytically inactive forms of Src kinases. These cells were unable to enter S phase in response to I’DGF, and we therefore conclude that Src family tyrosine kinases are required in order for the I’DGF receptor to transmit a mitogenic signal. It has previously been shown that the kinase activity of Src is negatively regulated by phosphorylation of tyr 527 in its carboxy-terminal tail. A kinase, Csk, that phosphorylates tyr 527 has recently been identified. We expressed Src in yeast to test the model that phosphorylation of tyr 527 represses activity by promoting intramolecular association between the tail and the SH2 domain. Inducible expression of Src in .S’. pombe caused cell death. Co-expression of Csk counteracted this effect. Src proteins mutated in the SH2 domain were as lethal as wild-type Src, but were insensitive to Csk. We interpret these results in favour of an SH2 domain : phosphorylated tail interaction repressing Src activity. However, we have also found that Src molecules containing mutations in the SH3 domain are not regulated by Csk. suggesting that the SH3 domain also functions in the intramolecular regulation of Src activity.