Mouse-liver gamma-glutamyl hydrolase (GH) is a lysosomal endopeptidase with an acid pH optimum that is activated by sulfhydryl compounds and preferentially hydrolyzes the most proximal gamma-glutamyl linkage of longer chain polyglutamates of folates and their analogues. We describe the cloning of this mouse lysosomal cDNA enzyme from liver GH mRNA in the form of two cDNA variants (1.295 and 1.268 kb in length) differing 14-fold (Variant I versus Variant II) in relative frequency that exhibited 5'-end heterogeneity and encoded alternate leader peptides. The 5' UTR in these variants also differs in length by 27 nucleotides. Otherwise, the ORF and 3' UTR in each case are the same. These cDNAs encode a protein in which the deduced amino acid sequence shares 78.9 and 69. 1% identity to rat and human GH sequences, respectively. Amino acid sequence comparisons among the three species identified three conserved Asn sites and two conserved Cys residues that may be sites of glycosylation and sulfhydryl compound activation, respectively. Variant I GH mRNA was more abundant than Variant II GH mRNA in all mouse tissues examined. Variant I GH mRNA levels were extremely high in salivary gland, moderately high in kidney, liver, lung, stomach and uterus, low in small intestine, brain and fetal liver and relatively rare in thymus, spleen and skeletal muscle. Abundance of GH mRNA among tumors varied from low to high, with no discernible correlation with their tissue of origin.