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Protein kinase C (PKC) ζ is a phospholipid-dependent serine/threonine kinase that appears to perform important cell signaling functions. Two forms of PKC ζ RNA, with different 5′ ends, have been reported. The major form (ζ) is expressed in most, if not all tissues, while the minor form (ζ′), which encodes the catalytic domain of the enzyme without most of its regulatory domain, is predominant in normal brain and certain rat prostate tumors. We report here the structure of the 5′ end of the rat PKC ζ gene, demonstrating that both forms of RNA can be transcribed from the same gene through the use of alternative promoters and splicing. In luciferase reporter constructs, progressive deletions of the PKC ζ and ζ′ 5′ flanking sequences yielded activities that were higher in the cell lines expressing endogenous PKC ζ and ζ′ RNAs, respectively. Also, multiple PCRs across different introns of the PKC ζ gene indicate that recent duplication of the gene or the existence of a closely related pseudogene in the rat genome are unlikely.