The regulatory sequence elements of the human muscle phosphofructokinase (HPFKM) p1 promoter from -655 to +78 were cloned and characterized. In the human cervical carcinoma cell line, HeLa S3, the HPFKM type C RNA initiated from a single predominant transcription initiation site and the HPFKM p1 promoter displayed transcriptional activity in transient transfection assays. The HPFKM p1 promoter region was shown to possess eight binding sites for the Sp1 transcription factor by DNase I footprinting and gel retardation analysis. The functional importance of these interactions was examined by transient transfection analysis in Drosophila SL2 and HeLa S3 cells. This analysis demonstrated that the HPFKM p1 promoter sequence between +12 and +78 retained Sp1-dependent transcriptional activity in Drosophila SL2 cells and retained promoter activity in HeLa S3 cells. These results suggest that the Sp1 binding site (site 8 between +12 and +21) immediately adjacent to the transcription initiation site represents an important regulatory element of this promoter at least in the context of the minimal HPFKM p1 promoter. However mutagenesis of the Sp1 site 8 demonstrated that, in the context of a larger HPFKM p1 promoter region containing Sp1 sites 1 to 7, it now contributed very little to the total promoter activity. Therefore it appears the Sp1 sites in the HPFKM p1 promoter display functional redundancy.