The parasitic protozoon Leishmania mexicana possesses an abundance of developmentally regulated cathepsin L-like cysteine proteinases expressed at highest levels in amastigotes. We recently characterised lmcpa, a single-copy gene encoding one such proteinase, LmCPa, which differs from other homologues by possessing a 3-amino-acid insertion at the amino terminal of the predicted mature proteinase. To investigate the role of LmCPa in L. mexicana, we used gene-targeting of promastigotes with hygromycin- and phleomycin-resistance markers to generate null mutants by disrupting sequentially both alleles of lmcpa. The promastigote null mutants did not differ significantly from wild-type L. mexicana in growth rate or morphology and could differentiate to metacyclics and the amastigote-like form, both of which could infect the J774G8 macrophage-like cell line. The null mutant amastigote-like form obtained from the J774G8 cells could also establish rump lesions in CBA mice. By these criteria, therefore, LmCPa appears to be non-essential although there is the possibility that LmCPa could be required during development in the sandfly, a stage not analysed here. The apparent redundancy of LmCPa in amastigotes may be due to the presence of other cysteine proteinases and has implications for the choice of candidate targets for rationally designed anti-leishmanial drugs.