Three peaks of tyrosine protein kinase activity (TK-I, TK-II and TK-III) can be resolved when the extract of rat spleen particulate fraction is subjected to DEAE-cellulose gradient chromatography. TK-I and TK-II, insensitive to both EGF and insulin, have been further purified by Sephacryl S200 gel filtration and characterized. TK-I has an apparent mR of 65000, by far prefers Mn2+ over Mg2+ as activator, can use GTP besides ATP as phosphate donor and is stimulated 2-3-fold by polylysine. TK-II, whose mR approximates 50000, is equally activated by Mg2+ and Mn2+, does not use GTP and is insensitive to polylysine. TK-I and TK-II can phosphorylate the synthetic peptide Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly (as well as its derivative with Orn in place of Arg), angiotensin II and poly(Glu, Tyr) 4:1 which exhibits different km values with TK-I and TK-II, (100 and 10 microM, respectively). When TK-I was incubated with [gamma-32P]ATP and MnCl2 a doublet of alkali-stable radiolabeled bands with molecular masses of 55 and 60 kDa were observed. Under identical conditions TK-II gives rise to a single alkali-stable radiolabeled band of 51 kDa, which may represent the autophosphorylation product of TK-II itself.