The major surface glycoprotein of Leishmania major is a zinc metalloproteinase of 63 kDa referred to as leishmanolysin or GP63, which is encoded by a family of seven genes. Targeted gene replacement was used to delete gp63 genes 1–6 encoding the highly expressed promastigote and constitutively expressed GP63. In the L. major homozygous mutants deficient in gp63 genes 1–6, there was no expression of GP63 as detected by reverse transcription–polymerase chain reaction (RT–PCR) or fluorescent staining in promastigotes from the procyclic stage (logarithmic growth phase). The remaining L. major gP63 gene 7 was shown to be developmentally regulated, as it was expressed exclusively in infectious metacyclic stage (late stationary growth phase) promastigotes and in lesion amastigotes. The gp63 genes 1–6-deficient mutants showed increased sensitivity to complement-mediated lysis. The sensitivity to lysis was greater in procyclics than in metacyclics when compared with the equivalent wild-type stages. Increased resistance of the mutant metacyclic promastigotes correlated with the expression of gp63 gene 7 and was restored to the same levels as wild-type promastigotes by transfection with gp63 gene 1. Thus, expression of GP63 is clearly involved in conferring resistance to complement-mediated lysis. The L. major GP63 1–6 mutants were capable of infecting mouse macrophages and differentiating into amastigotes. Similar levels of infection and subsequent intracellular survival were observed when mouse macrophages were infected in vitro with wild type, GP63 1–6 mutants and mutants transfected with gp63 gene 1. The GP63 1–6 mutants were capable of lesion formation in BALB/c mice and, thus, gp63 genes 1–6 do not play a role in the survival of the parasite within mouse macrophages. The role of gp63 genes 1–6 in parasite development within the sandfly vector was studied. GP63 1–6 mutants grew normally in the blood-engorged midgut of both Phlebotomus argentipes and P. papatasi. However, both wild-type and mutant promastigotes were lost after 2 days' growth in P. papatasi. The complete developmental pathway in P. argentipes was observed for wild-type promastigotes, GP63 1–6 mutants and mutants transfected with gp63 gene 1. Normal stage differentiation from amastigotes to procyclics, to nectomonads, to haptomonads and to infectious metacyclics was observed. Thus, the highly expressed promastigote forms of GP63, encoded by gp63 genes 1–6, do not appear to be required for nutrient utilization in the bloodmeal during the early stages of development in the sandfly or for midgut attachment and further development. gp63 1–6 genes do, however, play a major protective role against complement-mediated lysis when promastigotes are introduced into the mammalian host.