Low levels of hydrogen peroxide (H 2 O 2 ) are mitogenic to mammalian cells and stimulate the hyperphosphorylation of heterogeneous nuclear ribonucleoprotein C (hnRNP-C) by protein kinase CK1α. However, the mechanisms by which CK1α is regulated have been unclear. Here it is demonstrated that low levels of H 2 O 2 stimulate the rapid dephosphorylation of CK1αLS, a nuclear splice form of CK1α. Furthermore, it is demonstrated that either treatment of endothelial cells with H 2 O 2 , or dephosphorylation of CK1αLS in vitro enhances the association of CK1αLS with hnRNP-C. In addition, dephosphorylation of CK1αLS in vitro enhances the kinase’s ability to phosphorylate hnRNP-C. While CK1α appears to be present in all metazoans, analysis of CK1α genomic sequences from several species reveals that the alternatively spliced nuclear localizing L-insert is unique to vertebrates, as is the case for hnRNP-C. These observations indicate that CK1αLS and hnRNP-C represent conserved components of a vertebrate-specific H 2 O 2 -responsive nuclear signaling pathway.