We have cloned two mouse cDNAs encoding previously unidentified membrane-bound dipeptidases [membrane-bound dipeptidase-2 (MBD-2) and membrane-bound dipeptidase-3 (MBD-3)] from membrane-bound dipeptidase-1 (MBD-1) deficient mice (Habib, G.M., Shi, Z-Z., Cuevas, A.A., Guo, Q., Matzuk, M.M., and Lieberman, M.W. (1998) Proc. Natl. Acad. Sci. USA 95, 4859-4863). These enzymes are closely related to MBD-1 (EC 3.4.13.19), which is known to cleave leukotriene D4 (LTD4) and cystinyl-bis-glycine. MBD-2 cDNA is 56% identical to MBD-1 with a predicted amino acid identity of 33%. The MBD-3 and MBD-1 cDNAs share a 55% nucleotide identity and a 39% predicted amino acid sequence identity. All three genes are tightly linked on the same chromosome. Expression of MBD-2 and MBD-3 in Cos cells indicated that both are membrane-bound through a glycosylphosphatidyl-inositol linkage. MBD-2 cleaves leukotriene D4 (LTD4) but not cystinyl-bis-glycine, while MBD-3 cleaves cystinyl-bis-glycine but not LTD4. MBD-1 is expressed at highest levels in kidney, lung, and heart and is absent in spleen, while MBD-2 is expressed at highest levels in lung, heart, and testis and at somewhat lower levels in spleen. Of the tissues examined, MBD-3 expression was detected only in testis. Our identification of a second enzyme capable of cleaving LTD4 raises the possibility that clearance of LTD4 during asthma and in related inflammatory conditions may be mediated by more than one enzyme.