Casein kinase II (CKII) is a ubiquitous serine/threonine protein kinase with numerous key functions in cell metabolism and growth. The human CKII has a tetrameric structure; two catalytic subunits (alpha and alpha') form the holoenzyme together with two presumably regulatory subunits (beta). The gene encoding CKII subunit beta was isolated from human genomic DNA and analyzed for its primary structure using exclusively nonradioactive procedures. The gene was found to span 4.2 kilobase pairs and to be composed of seven exons. Exon sizes range from 76 (exon 5) to 329 base pairs (bp) (exon 1), intron sizes from 145 (intron V) to 965 bp (intron II). All exon-intron junctional sequences conform to the canonical GT-AG rule. Primer extension analysis determined three transcription initiation sites, at 951, 919, and (minor) 840 bp upstream of the translation start site. The translation start is located early in the second exon; exon 1 is untranslated. The 3'-cleavage/polyadenylation signal sequence (AA-TAAA) is in the last exon at position 4173 bp relative to the first transcription initiation site. The coding sequence for CKII beta comprises 648 nucleotides identical to the published CKII beta-cDNA sequence (Jakobi, R., Voss, H., and Pyerin, W. (1989) Eur. J. Biochem. 183, 227-233). The upstream promoter region of the CKII beta gene contains multiple potential gene regulatory sequence elements, noticeable DNA structures, and the characteristics of a housekeeping gene (more than one transcription initiation site, lack of a TATA-box, presence of a CpG island, occurrence of multiple GC boxes and of nonstandard positioned CCAAT boxes). The CKII beta gene promoter shares common features with that of mammalian protein kinases and is closely related to the regulatory subunit gene promoter of cAMP-dependent protein kinase.