Membrane-associated calcium-independent phospholipase Aγ7 (iPLA2γ) contains four potential in-frame methionine start sites (Mancuso, D.J. Jenkins, C.M. & Gross, R.W. (2000) J. Biol. Chem. 275, 9937-9945), but the mechanisms regulating the types, amount and subcellular localization of iPLA2γ in cells are incompletely understood. We now: (a) demonstrate the dramatic transcriptional repression of mRNA synthesis encoding iPLA2γ by a nucleotide sequence nested in the coding sequence itself; (b) localize the site of transcriptional repression to the most 5′ sequence encoding the iPLA2γ holoprotein; (c) identify the presence of nuclear protein constituents which bind to the repressor region by gel shift analysis; (d) demonstrate the translational regulation of distinct iPLA2γ isoforms; (e) identify multiple novel exons, promoters, and alternative splice variants of human iPLA 2γ; (f) document the presence of dual-competing subcellular localization signals in discrete isoforms of iPLA2γ; and (g) demonstrate the functional integrity of an N-terminal mitochondrial localization signal by fluorescence imaging and the presence of iPLA2γ in the mitochondrial compartment of rat myocardium. The intricacy of the regulatory mechanisms of iPLA2γ biosynthesis in rat myocardium is underscored by the identification of seven distinct protein products that utilize multiple mechanisms (transcription, translation and proteolysis) to produce discrete iPLA2γ polypeptides containing either single or dual subcellular localization signals. This unanticipated complex interplay between peroxisomes and mitochondria mediated by competition for uptake of the nascent iPLA2γ polypeptide identifies a new level of phospholipase-mediated metabolic regulation. Because uncoupling protein function is regulated by free fatty acids in mitochondria, these results suggest that iPLA2γ processing contributes to integrating respiration and thermogenesis in mitochondria.