SR-protein-specific kinase 1 (SRPK1) is first identified as a specific kinase for SR splicing factors. By RT-PCR of a conserved kinase domain, novel SR-protein-specific kinase clones were isolated from mouse brain. The cloned cDNAs encode a 106 kDa protein (648 amino acids, 92% identical to human SRPK1) and a 120 kDa protein (681 amino acids, 58% identical to human SRPK1). Therefore, they were designated mSRPK1 and mSRPK2, respectively. Northern blotting revealed the ubiquitous expression of mSRPK1 in all tissues examined and the tissue-specific expression of mSRPK2 in testis, lung, and brain. Both kinases phosphorylated SF2/ASF, a member of SR proteins in vitro and the phosphopeptide mappings were identical, indicating that these kinases phosphorylate the same site of SF2/ASF. Overexpression of mSRPK2 caused disassembly of cotransfected SF2/ASF and endogenous SC35. Our results indicate that SRPK family members may regulate the disassembly of the SR proteins in a tissue-specific manner.