We have examined the state of tyrosine phosphorylation of ligand-bound, internalized platelet-derived growth factor (PDGF) beta-receptors. Analysis by immunofluorescence staining of cells stimulated with PDGF-BB at 37 degrees C indicated colocalization of phosphotyrosine, PDGF beta-receptors, and PDGF-BB in endosome-like vesicles. Treatment of cells with an acidic buffer, which removed cell surface-bound PDGF-BB, led to a considerable decrease in phosphorylation and kinase activity of cell surface localized PDGF beta-receptors, but not of internalized receptors. Immunoprecipitations using antisera against phosphotyrosine and the PDGF beta-receptor from metabolically labeled cells showed that a major fraction of the tyrosine-phosphorylated pool of receptors were still accessible to the acid buffer treatment after 10 min of incubation of the cells at 37 degrees C. Under these conditions, about 20-25% of the total pool of tyrosine-phosphorylated receptors were intracellular, since they remained tyrosine phosphorylated after the acid buffer treatment. A considerable pool of tyrosine-phosphorylated, internalized receptors, after 10 min of incubation of the cells at 37 degrees C, could also be detected by immunoblotting analysis, using antisera against the PDGF beta-receptor and phosphotyrosine. Analysis by in vitro kinase assays of immunoprecipitated PDGF beta-receptors, obtained from PDGF-BB-stimulated cells different times after acid wash, showed that the internalized receptors retained kinase activity. These data suggest that a pool of internalized PDGF beta-receptors remain active and may participate in signalling a considerable time after internalization.