Insulin-like growth factor binding protein-2 (IGFBP-2), the predominant IGFBP in the fetal circulation and an induced protein during several types of malignancies, belongs to a family of structurally related proteins that bind the mitogens, IGF-1 and IGF-2. The present study focused on functional analysis of the 5 '-flanking region (approximately 1.3 kb) of the IGFBP-2 gene to identify nuclear factors that mediate hepatic transcription of this gene. Luciferase (LUC) reporter constructs containing progressive deletions of 5'-flanking DNA and the intact promoter of the porcine IGFBP-2 gene were examined for functional activity by transient transfection of human HepG2 liver cells. LUC activity of the transfected reporter gene driven by the IGFBP-2 promoter and flanking sequences to -1397 (numbering relative to initiation codon at +1) was 22-fold higher than that of promoterless parent LUC vector. This activity was decreased by 60% with deletion of sequences to -874 bp, and dropped to basal levels with further truncation to -764 bp. The region between -874 and -765 bp (110 bp) functioned as a potent stimulator of heterologous SV40 promoter activity (110 bp/SV40-LUC construct) and was found to contain two noncontiguous basic helix-loop-helix (bHLH) transcription factor binding motifs (E-boxes [CAN NTG]: CACCTG and CAAATG). In electrophoretic mobility shift assays, nuclear proteins prepared from HepG2 cells formed two complexes (C1, C2) with double-stranded oligonucleotides containing either HLH sequence, mutations of which resulted in loss of complex formation. Southwestern blot analysis identified an HepG2 nuclear protein with molecular mass of 48 kDa, similar to that of the bHLH transcription factor AP-4, which bound the CACCTG motif. Cotransfection of HepG2 cells with the 110-bp/SV40-LUC construct and an expression vector encoding human AP-4 increased IGFBP-2 fragment-dependent SV40 promoter activity by 16-fold. This AP-4-mediated stimulation was lost following block mutation of both bHLH motifs within the IGFBP-2 110-bp fragment. Results demonstrate the functional importance of sequences upstream of the promoter in IGFBP-2 gene transcription and identify a novel mechanism by which bHLH proteins potentially may affect cell proliferation and differentiation via induction of IGFBP-2 synthesis.