We have recently shown that three types (A,B, and C) of mRNA species are transcribed from a single gene encoding human muscle phosphofructokinase (hPFK-M) through alternative splicing [Nakajima et al., Biochem. Biophys. Res. Commun. 166 (1990) 637-641]. To determine its complete structure and elucidate the mechanism of alternative RNA splicing, we isolated the hPFK-M gene, which spans about 30 kb, and contains 24 exons. Transcription start points were observed for both exon 1 and exon 2 by S1 nuclease protection assay and primer extension. Motifs of an Sp1-binding site were observed in the upstream region of exon 1 (promoter 1). A TATA-box-like sequence and a CAAT-box-like sequence were identified in the upstream region of exon 2 (promoter 2). Reporter assay revealed that the promoter 1 region was functional both in HeLa cells and myoblastic clonal cells, and that the promoter 2 region was active only in myoblastic cells. Motifs of M-CAT known as a muscle-specific enhancer, were observed in the promoter 2 region. These results indicated that the hPFK-M gene contains at least two promoter regions, facilitating the expression of the heterogeneous gene transcripts in a cell-type-specific manner.