Pyruvate carboxylase (PC), a nuclear-encoded mitochondrial enzyme, catalyzes the conversion of pyruvate to oxaloacetate. Oxaloacetate is a substrate for multiple intermediary metabolic pathways including gluconeogenesis and lipogenesis. Inborn errors of PC metabolism cause lactic acidosis and mental retardation, and abnormal PC expression in uncontrolled diabetes mellitus may exacerbate post-absorptive hyperglycemia. To better understand the structure and function of PC in both health and disease, we have characterized the genomic structure of human PC. Five clones from several human genomic libraries encompassing the regulatory and coding region of human PC were isolated using human PC cDNA fragments as probes. Within the coding region there are 19 introns whose intron/exon junctions have been mapped. The coding region spans approximately 30 kb of the genomic gene. Utilizing a fragment of the gene as a probe for fluorescent in situ hybridization, the gene has been localized to human chromosome 11q13. The transcription start site was identified by reverse transcription and DNA amplification and found to extend 135 nucleotides upstream from the start methionine. Approximately 1.2 kb of the 5′ regulatory region has been sequenced. The regulatory region contains a TATA-less promoter with a CAAT site located 40 nucleotides upstream of the transcription start site. Knowledge of the genomic structure of PC will facilitate studies of the regulation and structure of PC. (Part of this work was supported by NIH grant HD32434.)