The phosphorylatlon of proteins on tyrosine in vivo and in vitro was examined in ST3 cells stimulated by platelet-derived growth factor (PDGP) and transformed by polyoma middle T antigen (MT@) by using an antibody directed against phosphotyrosine (P-tyr). lwo common events were observed upon PDGF stimulation or MTAg transformation of cells: the appearance in the immunoprecipitates of an 85 kd phosphoprotein, and Increased phosphatidyllnositol(PI) klnase activity. In PDGF-stimulated cells, the 85 kd phosphoprotein and PI kinaee activity appeared rapidly, within 1 min of growth factor addition. The PI kinase activity and 85 kd phosphorylation were also increased in anti-Ptyr immunoprecipitates from cells transformed by v-fms and vs &, but not by SV40 T antigen. The presence of the tyrosine-phosphorylated 85 kd protein correlated with PI kinase activity during several purification steps. These msuits suggest that the 85 kd phosphoprotein, a putative PI kinase, is a substrate for both the PDGF receptor and MTAg/~ p80*‘~ tyrosine kinase activities.