cDNA clones for rat R-type pyruvate kinase and a genomic clone encoding both L- and R-type isozyme mRNAs were isolated. Their sequences were compared with that of the L-type isozyme cDNA to determine the sequences of mRNA and protein of the R-type isozyme and the organizations of the L- and R-type genes. Results showed that the R-type isozyme mRNA had an identical nucleotide sequence to that of the L-type except in the 5'-terminal region including the coding sequence and the length of the 3'-untranslated region. The sequence upstream of the 5th coding residue of the L-type was replaced by a 98-nucleotide coding sequence plus a 5'-untranslated region in the R-type isozyme. Therefore, the R-type subunit consists of 574 amino acids, which is 31 residues longer than the L-type at the amino terminus. The pyruvate kinase L gene is present as a single copy per haploid genome and is composed of 12 exons and 11 introns with a length of about 9.3 kilobase pairs. The first (exon R) and second (exon L) exons encode the 5'-terminal sequences specific for the R- and L-types, respectively. The remaining downstream exons encode a sequence common to both isozymes. The last exon contains the entire 3'-untranslated region, including several putative polyadenylation signals. Alternative use of these signals is reported to be responsible for generation of multiple mRNA species for the L-type, whereas the R-type uses only the first signal. The cap site is mapped 16 nucleotides upstream from the translation initiation site for the L-type, whereas multiple cap sites were suggested for the R-type. The canonical promoter of the TATA box was identified in the upstream sequence of exon L, but not in that of exon R. Instead, the 5'-flanking region of exon R contained another promoter sequence of the CAT box. Thus, we conclude that the L- and R-type isozymes of pyruvate kinase are produced from a single gene by use of different promoters.