Clinical expression of Hirschsprung disease (HSCR) requires the interaction of multiple susceptibility genes. Molecular genetic analyses have revealed that interactions between mutations in the genes encoding the RET receptor tyrosine kinase and the endothelin receptor type B (EDNRB) are central to the genesis of HSCR. We have established two locus noncomplementation assays in mice, using allelic series at Ednrb in the context of Ret kinase-null heterozygotes, to understand the clinical presentation, incomplete penetrance, variation in length of aganglionic segment, and sex bias observed in human HSCR patients. Titration of Ednrb in the presence of half the genetic dose of Ret determines the presentation of an enteric phenotype in these strains, revealing or abrogating a sex bias in disease expression depending on the genotype at Ednrb. RET and EDNRB signaling pathways are also critical for the normal development of other tissues, including the kidneys and neural crest-derived melanocytes. Our data demonstrate that interaction between these genes is restricted to the enteric nervous system and does not affect renal, coat color, and retinal choroid development. C omplex inheritance minimizes the impact of genetic variation and suppresses the potential expression of deleterious phenotypes by requiring the combined influence of mutant alleles at multiple loci to reveal a disease phenotype. Hirschsprung disease (HSCR) or aganglionic megacolon is a complex trait requiring the interaction of multiple genes for disease expression. Mutations in eight genes have already been identified in patients with HSCR and include RET, GDNF, NRTN, EDNRB, EDN3, ECE1, SOX10,. HSCR also displays several genetic hallmarks, including incomplete penetrance and pleiotropic effects of mutant genotypes, a marked sex difference in clinical expression, and variation in penetrance with extent of aganglionosis. Genetic and epigenetic modification of known mutations may provide a parsimonious explanation for these findings, suggesting that the majority of HSCR cases are likely to arise from the combined effects of multiple susceptibility genes. Although studies in mice have uncovered the disease phenotypes resulting from mutations within HSCR genes, we know little of the influence of variation in multiple genes or genetic background. Variants that abrogate a gene's capacity to moderate disease expression reveal interactions intrinsic to the associated trait. We have begun to dissect these interactions in enteric nervous system (ENS) development and HSCR and report specific genetic buffering interactions in ENS development and HSCR.HSCR is a congenital malformation with an incidence in the general population of 1͞5,000 live births (14, 15) and is characterized by an absence of neural crest (NC)-derived intrinsic ganglia along a variable length of the distal intestinal tract. The pathways mediated by the RET receptor tyrosine kinase and the G-protein-coupled endothelin receptor type B (EDNRB) are critical for the normal development of the ENS and ...