Two different cDNAs have been isolated and characterized from a rat kidney cDNA library. The two cDNA sequences are identical in the coding region and in the 144 bases upstream from the initiation codon but have alternate sequences (154 and 138 bases) at their 5' ends. Primer extension analysis on kidney mRNA reveals that both cDNAs are full-length and correspond to two mRNAs of nearly the same size (2142 and 2127 bases). Synthesis of two mRNAs with alternative 5' ends can be explained only by initiation at two separate promoters on the single rat gamma-glutamyl transpeptidase (GGT) gene. The alternate 5' end nucleotide sequences were used as probes to detect the corresponding mRNAs in several rat tissues. In the kidney, the expression of both RNAs was detected by in situ hybridization in the distal part of the proximal convolutions of the renal tubule. Northern blot analysis of kidney mRNAs reveals that the expression of both mRNAs increases from birth to the adult stage. Neither of these two transcripts is expressed in the liver or in seminal vesicles in which a larger mRNA (2.4 kilobase pairs) is transcribed from the same gene. Thus, two GGT mRNAs, initiated on two separate promoters on the single GGT gene, are expressed in the rat in a tissue-specific manner and coordinately regulated.