Transcription of the L-type pyruvate kinase (L-PK) gene is induced in response to increased carbohydrate metabolism in the liver. We have demonstrated previously that a segment of the 5'-flanking region of the L-PK gene between -183 and -96 is necessary and sufficient for the glucose response in primary hepatocytes. To explore the protein factors that are involved in carbohydrate regulation, we have performed mutational analyses and in vitro binding studies of this segment. Sequences critical for the glucose response were mapped from -171 to -124. This segment contains the consensus binding sites for two nuclear transcription factors: LF-A1 and MLTF. Both factors are capable of binding to the corresponding L-PK sites in vitro. Mutational and functional analyses indicated that LF-A1 is indeed involved in glucose induction of the L-PK gene. The PK MLTF-like site consists of two imperfect CACGTG motifs, the core binding site for a family of transcription factors related to c-myc. Unexpectedly, mutations in either motif that resulted in defective glucose stimulation retained in vitro binding to MLTF. Furthermore, an authentic MLTF binding site from the adenovirus major late promoter was not functionally interchangeable with the natural sequence. These data indicate that binding of MLTF, in presence of LF-A1, is not capable of supporting the glucose response. Conversion of either imperfect motif to CACGTG within the context of the mutations disrupting the opposite site restored the response to elevated glucose. Thus, the factor that recognizes the PK MLTF-like site and participates in mediating the carbohydrate response of the L-PK gene appears to be a member of the c-myc family distinct from MLTF.