Fructose 6-phosphate 2-kinase/fructose 2,6-bisphosphatase catalyzes the synthesis and degradation of fructose 2,6-bisphosphate, which is the most potent activator of glycolysis. We have shown previously the occurrence of a partial cDNA (RB7) encoding the catalytic core domain of a novel brain-type isozyme. To elucidate the full-length sequence of RB7 cDNA, we have carried out cDNA cloning using the 3'- and 5'-rapid amplification cDNA ends method and have isolated eight isoforms from rat brain. The cDNA sequences encoding the 5'-untranslated region, the amino-terminal domain, and the catalytic core domain were identical among all the isoforms. However, heterogeneity of the carboxyl-terminus was found by sequence analysis. This heterogeneity was shown not to have resulted from transcription of multiple genes, as Southern blot and genomic sequence analysis revealed that the gene was a single copy in the rat genome. It is likely that these transcripts represent splice variants of the gene. High-level expression of the gene was also observed by northern blot analysis in skeletal muscle. However, the pattern of alternative splicing was different from that of brain, and only four isoforms were detected in skeletal muscle.