PYST2 is a member of a structurally homologous subfamily of MAP kinase phosphatases. A computer-based analysis of the PYST2 locus revealed that it harbors two alternative open reading frames promoted by two conserved promoter regions. Using Northern blot analysis and reverse transcription-polymerase chain reaction followed by sequencing and alignment of the products, we confirmed the existence of two mRNAs that were transcribed from this genomic region. Western blot analysis indicated that these transcripts were translated, Functional bioinformatic analysis of both transcripts revealed that exon 2 exists in only one of the PYST2 transcripts, designated PYST2-L, and has the consensus elements of the phosphatase catalytic domain (PCD). We found that the translation from the PYST2-L transcript starts 46 codons upstream from the (already-known) PYST2 5′ sequence. Furthermore, the existence of three PYST2-L transcripts was indicated. These transcripts differ only in their 5′ untranslated regions (5′UTRs). Unlike PYST2-s, the other mRNA (PYST 2-S) is devoid of any known PCD. Analysis of the predicted Pyst2-S protein revealed the presence of the vertebrate metallothionein signature I, the mammalian defensin, and the zinc-containing alcohol dehydrogenase motifs. These motifs might confer on this protein the ability to sense changes in the cellular environment. From these and previous results, we speculate that Pyst2-S may function as a negative regulator of Pyst2-L.