The protein tyrosine kinase expressed by the protooncogene c-fgr is phosphorylated and down-regulated in vitro by the c-Src kinase (CSK). CSK catalyzed phosphorylation affects Tyr-511 of c-Fgr, homologous to Tyr-527 of c-Src and it prevents the autophosphorylation normally occurring at c-Fgr Tyr-400, homologous to c-Src Tyr-416. Polylysine, histones H1 and H2A and other polycationic proteins on the other hand stimulate c-Fgr activity while promoting enhanced autophosphorylation of both Tyr-400 and Tyr-511. Once phosphorylated at Tyr-511 and down-regulated by CSK, c-Fgr is no more susceptible to polylysine stimulation. Previous autophosphorylation (at Tyr-400) reduces c-Fgr susceptibility to down-regulation by CSK, although Tyr-511 can be still phosphorylated by it. If a more exhaustive autophosphorylation (of both Tyr-400 and Tyr-511) is performed in the presence of polylysine, c-Fgr becomes totally insensitive to CSK down-regulation. These data support the concept that down-regulation of c-Fgr by Tyr-511 phosphorylation is prevented if Tyr-400 is also phosphorylated and they are consistent with an outcompetition of phospho-Tyr-511 from the Src homology 2 domain by phospho-Tyr-400, which, in c-Fgr, is surrounded by an amino acid sequence divergent from that of the other Src-related protein tyrosine kinases.