Protein tyrosine phosphatases comprise a large family of enzymes that are involved in the control of cellular tyrosine phosphorylation. We have used lambda phage analysis to elucidate the complete genomic structure of an intracellular member of this family, the murine MPTP-PEST gene. Eight overlapping lambda phage clones representing the MPTP-PEST locus were isolated from a 129/sv mouse genomic library. The gene spans over 90 kb of the mouse genome and is composed of 18 exons, 10 of which constitute the catalytic phosphatase domain. Detailed comparison of the position of intron/exon boundaries of the phosphatase domain of MPTP-PEST to those of several other protein tyrosine phosphatases indicates that the MPTP-PEST catalytic domain contains additional exons as a consequence of the insertion of novel introns. In addition, this analysis reveals a strong conservation of the genomic organization within the catalytic domain of the protein tyrosine phosphatase gene family. Finally, fluorescence in situ hybridization with MPTP-PEST genomic DNA refines the map position of MPTP-PEST to mouse chromosome 5A3 to B. This result is in agreement with the previous mapping of the human PEST gene to chromosome 7q11.23, a region of synteny with the centromeric portion of mouse chromosome 5.