ABSTRACT 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 D 4 (LTD 4 ) 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 D 4 (LTD 4 ) 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 LTD 4 raises the possibility that clearance of LTD 4 during asthma and in related inflammatory conditions may be mediated by more than one enzyme.