Abstract Phosphoinositide 3-kinase γ is preferentially expressed in leukocytes. PI3Kγ is activated by βγ subunits of heterotrimeric G-proteins, which thus link seven transmembrane helix receptor activation to phosphatidylinositol (3,4,5)-trisphosphate production. Here we describe the molecular cloning of the murine PI3Kγ cDNA, the PI3Kγ gene structure, its chromosomal assignment and the analysis of promoter activity. The mouse cDNA shares 86% identity to its pig and human orthologues at the nucleotide level. The MmPI3Kγ gene spans approximately 30 kb and comprises 11 exons. RACE-PCR indicated the presence of multiple start sites generating 5′ UTRs with different lengths, the longest being 874 bp. The putative promoter region contains no TATA box but several putative binding sites for hematopoietic specific transcription factors. A 1200 bp long sequence upstream the first transcription start site was found to possess tissue specific promoter activity. Deletion constructs revealed two contiguous regions, with activator function, ranging from positions −139 to −557, and with inhibitory function, ranging from positions −557 to −892. FISH analysis revealed that the MmPI3Kγ is located on chromosome 12 band B and that the human orthologue is positioned on chromosome 7q22.2–22.3. In spite of some differences in the ATP-binding site, recombinant murine PI3Kγ protein is equally sensitive to wortmannin as its human counterpart. This suggests that mouse models will provide reliable results in the assessments of novel PI3Kγ inhibitors.