Three C-terminal sequences (VSIPVV, VCKWT, and LTLLS) of rat heart-type fructose-6-phosphate 2-kinase/fructose-2,6-bisphosphatase have been reported. To elucidate the mechanism of generating the heterogeneity of the enzyme, we carried out reverse transcriptase PCR using three pairs of specific primers, The existence of mRNAs encoding VSIPVV and LTLLS was con firmed but not that of VCKWT. In addition to these cDNAs, we found four novel mRNAs that encode the C-termini of the enzyme. The genomic sequence reveals that the heterogeneity is generated by alternative splicing of exon 15 and four novel exons (exon 16a-d).