Two new human cDNAs, designated phclk2 and phclk3, which have a high identity to the cDNA of the human protein kinase clk, were characterized. Typical features of hclk2 and hclk3 proteins are non-homologous N-terminal regions and the presence of the C-terminal protein kinase domain, which is characteristic for serine/threonine-type kinases. We also identified the differentially spliced forms phclk2(139) and phclk3(152) with deletions of 88 and 97 nt, respectively, which lead to changes in the open reading frames. hclk2(139) and hclk3(152) proteins do not possess a protein kinase domain and are nearly identical to the N-terminal regions of the above-mentioned protein kinases. We verified that differentially spliced variants also exist for hclk1 as well as for a mouse clk protein kinase. It was shown that shorter and longer alternatively spliced mRNAs co-exist in different human tissues. According to Southern analysis, hclk2 and hclk3 appear to be specified by single copy genes. The genes for hclk2 as well as for hclk3 were localized to human chromosomes 1 and 15, respectively.