Polyclonal antibodies have been raised against two synthetic peptides reproducing the 48-64 and 353-369 sequences of CSK, a protein tyrosine kinase implicated in the down-regulation of src-related protein kinases. Both antibodies specifically recognize recombinant CSK and a CSK-related 49 kDa protein tyrosine kinase present in spleen but they do not cross-react with purified TPK-IIB, a spleen protein tyrosine kinase sharing with CSK catalytic activity toward src kinases and incapability to autophosphorylate. CSK and TPK-IIB once resolved from each other by heparin-Sepharose affinity chromatography, display opposite specificities toward synthetic peptides reproducing the sequences around the main phosphoacceptor residues of pp60c-src, namely Tyr-416 and Tyr-527. These data support the view that TPK-IIB and CSK may exert opposite effects on the activity of src-related protein tyrosine kinases.