A DNA fragment containing 1.5 kb of the 5'-flanking region of the human ubiquitous PFKFB3 gene, coding for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase, was cloned and its promoter activity was examined. The 5' flanking region contains a TATA box-like and GC-rich sequences, yielding several potential Specific protein (Sp-1) and activator protein (AP)-2 binding sites. Putative regulatory motifs for E-box, nuclear factor (NF)-1 and progesterone response element were also found by computer assisted analysis. Transient expression assays of truncated promoter-reporter constructs in HeLa cells showed that this gene is induced by phorbol esters (PDB) and cyclic-AMP-dependent protein kinase signal activation. Furthermore, the genomic organization of the PFKFB3 gene is reported. This gene spans more than 26 kb containing at least 16 exons that accounts for the two reported isoforms, inducible and ubiquitous, generated through alternative splicing of exon 15.