PTPN6, a protein tyrosine phosphatase with two Src homology-2 domains, is expressed predominantly in cells of the hematopoietic lineage. In addition, cDNAs encoding isoforms of the enzyme differing from the latter at their N-terminal have been detected in normal and transformed epithelial cells. To understand further the regulation and expression of the PTPN6 gene, we have isolated and characterized overlapping human genomic lambda clones containing the entire locus. The gene, located on chromosome 12, consists of 17 exons spanning 17 kb of DNA. Three nonhematopoietic PTPN6 transcripts were identified in a variety of cell lines and were shown to be transcribed from a common promoter. Alternate splicing and exon skipping result in transcripts encoding the two SH2 domains and minor transcripts from which part of or the entire N-terminal SH2 domain coding sequences are missing. The hematopoietic form of PTPN6 transcript is initiated at a downstream promoter separated by 7 kb from the first. This promoter is active exclusively in cells of the hematopoietic lineage. Characterization of the 5' ends of the PTPN6 mRNAs by RT-PCR and analysis of the flanking genomic sequences identified putative initiation sites within the two promoters.