Demodex musculi, a prostigmatid mite that has been reported infrequently in laboratory mice, has been identified with increasing frequency in contemporary colonies of immunodeficient mice. Here we describe 2 episodes of D. musculi infestation with associated clinical signs in various genetically engineered mouse strains, as well as treatment strategies and an investigation into transmissibility and host susceptibility. The first case involved D. musculi associated with clinical signs and pathologic lesions in BALB/c-Tg(DO11.10)Il13(tm) mice, which have a defect in type 2 helper T cell (Th2) immunity. Subsequent investigation revealed mite transmission to both parental strains (BALB/c-Tg[DO11.10] and BALB/c-Il13(tm)), BALB/c-Il13/Il4(tm), and wild-type BALB/c. All Tg(DO11.10)Il13(tm) mice remained infested throughout the investigation, and D. musculi were recovered from all strains when they were cohoused with BALB/c-Tg(DO11.10)Il13(tm) index mice. However, only Il13(tm) and Il13/Il4(tm) mice demonstrated persistent infestation after index mice were removed. Only BALB/c-Tg(DO11.10)Il13(tm) showed clinical signs, suggesting that the phenotypic dysfunction of Th2 immunity is sufficient for persistent infestation, whereas clinical disease associated with D. musculi appears to be genotype-specific. This pattern was further exemplified in the second case, which involved NOD.Cg-Prkdc(scid)Il2r(tm1Wjl)/SzJ (NSG) and C;129S4 Rag2(tm1.1Flv) Il2rg(tm1.1Flv)/J mice with varying degrees of blepharitis, conjunctivitis, and facial pruritis. Topical amitraz decreased mite burden but did not eliminate infestation or markedly ameliorate clinical signs. Furthermore, mite burden began to increase by 1 mo posttreatment, suggesting that topical amitraz is an ineffective treatment for D. musculi. These experiences illustrate the need for vigilance regarding opportunistic and uncommon pathogens in rodent colonies, especially among mice with immunologic deficits.