Glucose-regulated transcription of the L-type pyruvate kinase (L-PK) gene is mediated through its glucose response element (G1RE/L4 box) composed of two degenerated E-boxes. Upstream stimulatory factor (USF) is a component of the transcriptional glucose response complex built up on the G1RE. Cooperation of the G1RE with the contiguous binding site (L3 box) for the orphan nuclear receptor hepatocyte nuclear factor 4 (HNF4) has also been suggested. We compared by transient transfection assays the effects of USF2a and other basic helix-loop-helix leucine zipper (bHLH-LZ) factors (TFE3, c-Myc, SREBP/ADDl) on the activity and glucose responsiveness of a minimal L-PK promoter directed by oligomerized glucose response units (L4L3 boxes). We found that:(i) although USF2a is intrinsically a moderate transcriptional activator, it has a strong stimulatory effect on the activity of the L4L3-based reporter construct in hepatocyte-derived cells and interferes with the glucose responsiveness;(ii) despite its potent ability as a transactivator, TFE3 alone is barely active on the G1RE in hepatocyte-derived cells;(iii) TFE3 as USF2a acts synergistically with HNF4 and abolishes glucose responsiveness of the promoter when overexpressed;(iv) in contrast, overexpression of HNF4 alone stimulates activity of the promoter without interfering with glucose responsiveness;(v) SREBP/ADDl has a very weak activity on the L4L3 elements, only detectable in the presence of HNF4, and c-Myc does not interact with the G1RE of the L-PK promoter. Our studies indicate that different bHLH-LZ transcription factors known to recognize CACGTG-type E-boxes are not equivalent in acting through the L-PK glucose response element, with USF proteins being especially efficient in hepatocyte-derived cells.