In species as diverse as yeast and mammals, peptide:N-glycanase (PNG1 in yeast; Ngly1 in mouse) is believed to play a key role in the degradation of misfolded glycoproteins by the proteasome. In this study, we report the genomic organization and mRNA distribution of the mouse Ngly1. Mouse Ngly1 spans 61kb and is composed of 12 exons, the organization of which is conserved throughout vertebrates. Comparison of the mouse and human genomic sequence identifies a conserved gene structure with significant sequence similarity extending into introns. A 2.6kb Ngly1 message was detected in all mouse tissues examined, with the highest abundance in the testis. In addition, a lower molecular weight transcript of 2.4kb was detected in the testis. From analysis of dbESTs the alternative transcript of Ngly1 is predicted to be present in the human placenta. Given the key role Ngly1 plays in glycoprotein degradation, we predict that Ngly1 may be a contributing factor in "disease" susceptibility. To begin to address this question, we used radiation hybrid mapping to localize mouse Ngly1 to chromosome 14 and the human orthologue to chromosome 3 with a strong link with known genes.