Tyrosine kinases and phosphatases are regulators of the steady-state levels of phosphotyrosine proteins and, in this way, are key players in determining the functional state of the cell. As a unique member of the protein tyrosine phosphatase (PTP) superfamily, osteotesticular PTP (OST-PTP) is a receptor protein whose expression is highly regulated during osteoblast differentiation and in response to modulators of bone remodeling such as parathyroid hormone and vitamin D3. To explore the molecular mechanisms and signaling pathways important in the regulation of this gene, we characterized the structural organization of the mouse OST-PTP cDNA and gene and determined its chromosomal localization. The mouse cDNA is approximately 5.5 kb including 5.1 kb of coding sequence, 315 bp 5' UTR and 102 bp 3' UTR. It is expressed as a single approximately 5.8 kb transcript in day 8 differentiated MC3T3 osteoblasts. Although highly homologous to the rat OST-PTP cDNA, the mouse cDNA possesses a 74 bp insert in the 5' UTR which contains several potential transcription factor binding sites such as AP-2 and NFkappaB. The mouse OST-PTP (mOST-PTP) gene is a single copy gene encompassing 35 exons and spanning only 20.65 kb. As such, it is the smallest gene of the characterized receptor PTP genes. This is due to the lack of large introns and the conserved spatial organization of exons which encode specific protein motifs in the mOST-PTP molecule. Sequence analysis of the putative mOST-PTP promoter revealed basal elements as well as many potential cis-acting regulatory elements with relevance to gene regulation in bone. Of particular interest is the single osteoblast specific element known as osteocalcin specific element 2 (OSE2) found at position -1867, as well as numerous VDRE and NFkappaB sites found throughout the promoter and the 5' UTR. Fluorescence in situ hybridization studies have shown that mOST-PTP localizes to mouse chromosome 1, region F-G which is syntenic to the segment of human chromosome 1q32-33. This characterization of the mOST-PTP cDNA and gene will facilitate future experiments exploring the mechanisms of regulation of this phosphatase during osteogenesis.