AbstractHirschsprung disease (distal intestinal aganglionosis, HSCR) is a multigenic disorder with incomplete penetrance, variable expressivity, and a strong male gender bias. Recent studies demonstrated that these genetic patterns arise because gene interactions determine whether enteric nervous system (ENS) precursors successfully proliferate and migrate into the distal bowel. We now demonstrate that male gender bias in the extent of distal intestinal aganglionosis occurs in mice with Ret dominant‐negative mutations (RetDN) that mimic human HSCR. We hypothesized that male gender bias could result from reduced expression of a gene already known to be essential for ENS development. Using quantitative real‐time polymerase chain reaction (PCR) we demonstrated reduced levels of endothelin converting enzyme‐1 and endothelin‐3 mRNA in the male mouse bowel at the time that ENS precursors migrate into the colon. Other HSCR‐associated genes are expressed at comparable levels in male and female mice. Testosterone and Mullerian inhibiting substance had no deleterious effect on ENS precursor development, but adding EDN3 peptide to E11.5 male RetDN heterozygous mouse gut explants in organ culture significantly increased the rate of ENS precursor migration through the bowel. Developmental Dynamics 236:106–117, 2007. © 2006 Wiley‐Liss, Inc.