The LmcDNA16 locus of Leishmania major contains three highly related genes HASPA1, HASPA2 and HASPB, encoding hydrophilic, acylated surface proteins and a tandem pair of unrelated sequences, SHERP1 and SHERP2, coding for a small, hydrophilic protein that localizes to the endoplasmic reticulum and outer mitochondrial membrane. Differential regulation of these genes results in expression of a subset of the HASP proteins and SHERP only in infective stage parasites. To assess the contribution of these molecules to parasite virulence, the diploid LmcDNA16 gene locus has been removed by targeted gene deletion. Homozygous null mutants have precise deletions of both alleles and exhibit no HASP or SHERP expression. They are at least as virulent as wild-type parasites in macrophage invasion and intracellular survival assays, both in vitro and in vivo. Conversely, null mutants engineered to overexpress the entire LmcDNA16 gene locus are unable to survive within the intramacrophage environment despite their differentiation into infective metacyclic parasites. Both null and overexpressing null parasites show increased sensitivity to complement-mediated lysis, suggesting perturbation of their surface architecture. Avirulence in overexpressing parasites correlates with selective depletion of a specific lipid species, decreased expression of the major surface glycoprotein GP63, but no significant downregulation of the glycoconjugate lipophosphoglycan.