Genomic clones carrying the entire rat true tissue kallikrein gene have been isolated and characterized. Southern blot analysis revealed the presence of a unique gene for true tissue kallikrein in the rat haploid genome. The true tissue kallikrein gene was composed of 5 exons and 4 introns, its length being about 4.5 kilobase pairs. We also isolated the rat renal kallikrein cDNA which was expressed in the kidney. The exon sequences of the gene were identical with the corresponding sequences of the kallikrein cDNA. The rat true tissue kallikrein gene was distinct from two rat kallikrein-related genes expressed in the kidney (Chen, Y. et al. (1988) Biochemistry 27, 7189-7196). Primer extension analysis indicated that two identical transcription-initiation sites were used in both the kidney and submandibular gland, although expression of the true tissue kallikrein in the submandibular gland was much higher than that in the kidney. The 3'-noncoding region of this gene contained the polyadenylation signal, AATAAA, observed in the rat renal kallikrein cDNA. The 5'-flanking region was sequenced up to about 800 base pairs, upstream of the cap sites. CATCT, GGGCGG, and TTTAAA boxes were found as common signals required for eukaryote gene expression within about 100 base pairs-upstream of the cap sites.