Transcription of hepatic L-type pyruvate kinase (L-PK) gene is cell type-specific and is under the control of various nutritional conditions. The L-PK gene contains multiple cis-regulatory elements located within a 170-bp upstream region necessary for these regulations. These elements can synergistically stimulate L-PK gene transcription, although their mechanisms are largely unknown. Because nuclear factor (NF) 1 family members bind to specific cisregulatory elements known as L-IIA and L-IIB and hepatocyte nuclear factor (HNF) 1␣ binds to the adjacent element L-I, we examined the functional and physical interactions between these two transcription factors. Reporter gene assay showed that these two factors synergistically activated the L-PK promoter containing the 5-flanking region up to ؊189. Although two NF1-binding sites are required for the maximum synergistic effect of NF1 family members with HNF1␣, significant functional interaction between the two factors was observed in the L-PK promoter containing two mutated NF1-binding sites and also in the promoter containing only the HNF1␣-binding site, raising the possibility that NF1 proteins function as HNF1␣ co-activators. Chromatin immunoprecipitation assay revealed that both NF1 proteins and HNF1␣ bound to the promoter region of the L-PK gene in vivo. In vitro binding assay confirmed that NF1 proteins directly interacted mainly with the homeodomain of HNF1␣ via their DNA-binding domains. This interaction enhanced HNF1␣ binding to the L-I element and was also observed in rat liver by co-immunoprecipitation assay. Thus, we conclude that cooperative interaction between NF1 family members and HNF1␣ plays an important role in hepatic L-PK transcription.Pyruvate kinase (ATP, pyruvate 2-O-phosphotransferase; PK) 2 is an important regulatory enzyme in the glycolytic pathway. Four PK isozymes are known to exist in mammals and are designated as L, R, M 1 , and M 2 types (1). They are produced from two genes, PKL and PKM, by alternative use of two promoters and mutually exclusive alternative splicing, respectively (2-4). The L-type isozyme (L-PK) is expressed in a tissue-specific manner; this form is expressed primarily in hepatic parenchymal cells and is also present in pancreatic ␤ cells, kidney, and small intestine (1). Hepatic L-PK gene expression is also transcriptionally controlled positively by glucose in the presence of insulin and negatively by polyunsaturated fatty acids or glucagon via cyclic AMP (5-8). These regulations of the L-PK gene transcription are mediated via the multiple cis-regulatory elements located within a 170-bp upstream region from the transcription start site, as shown in Fig. 1 (8 -10). We have named these elements L-I (Ϫ94 to Ϫ76), L-II (Ϫ149 to Ϫ126), and L-III (Ϫ170 to Ϫ150) (9). In addition, another group has identified a weak negative element (Ϫ116 to Ϫ99) between L-I and L-II (10). Although this element has been named L2, we will refer to this as L-IIA and L-II as L-IIB hereafter. Although L-IIB is considered to be a regulatory element...