Phosvitin/casein kinase type II is an ubiquitous, highly conserved enzyme consisting of subunits alpha, alpha' and beta. Subunit beta, presumably serving regulatory functions, was prepared from human placenta and the amino acid sequence of a protease digestion peptide was determined. The deduced nucleotide sequence was employed for the synthesis of a mixture of 20mers as a hybridization probe to screen a lambda gt10 HeLa cell cDNA library for clones encoding subunit beta. A full-length clone consisting of 1013 bp was isolated and the sequence of both strands determined. The deduced amino acid sequence of the largest open reading frame encodes 215 amino acid residues predicting a maximum Mr of 24925. The nucleotide sequence of the human beta subunit shows a similarity of 75% to that of Drosophila melanogaster and the deduced amino acid sequence a similarity of 88% and 97% to that of D. melanogaster and bovine lung, respectively. No further protein sequence currently known exhibits any notable similarity. Using a 537-bp restriction fragment of the cDNA clone representing 80% of the coding region, a 1-kb subunit-beta transcript was detected by Northern hybridization in total RNA prepared from human epithelial cells and placenta as well as from bovine heart suggesting a single transcript of the beta-subunit gene and also demonstrating high similarity also between the human and bovine nucleotide sequences. The same restriction fragment was used as the probe to indicate in Southern hybridizations that the corresponding genomic DNA contains at least one intron of roughly 2.5 kb in length and presumably is a single copy gene.