The majority of adenovirus serotypes utilize the coxsackievirus-adenovirus receptor (CAR) for virus-host cell attachment, but subgroup B and subgroup D (adenovirus type 37 [Ad37]) viruses recognize CD46. CD46 is a ubiquitously expressed receptor that serves as a cofactor for the inactivation of the complement components C3b and C4b, and it also serves as a receptor for diverse microbial pathogens. A reported consequence of CD46 engagement is a reduced capability of human immune cells to express interleukin-12 (IL-12), a cytokine involved in both the innate and adaptive immune responses. Studies were thus undertaken to determine whether CD46-utilizing Ads alter the expression of proinflammatory cytokines. Subgroup B (Ad16 and -35) and Ad37, but not Ad2 or -5, significantly reduced IL-12 production by human peripheral blood mononuclear cells stimulated with gamma interferon (IFN-gamma) and lipopolysaccharide. IL-12 mRNA (p35 and p40 subunits) levels as well as other cytokine mRNA levels (IL-1alpha and -beta, IL-1Ra, and IL-6) were decreased upon interaction with CD46-utilizing Ads. Analysis of transcription factor activity required for cytokine expression indicated that CD46-utilizing Ads preferentially inhibited IFN-gamma-induced C/EBPbeta protein expression, consequently reducing its ability to form DNA complexes. Interference with IFN-gamma signaling events by CD46-utilizing Ads, but not CAR-utilizing Ads, reveals a potentially critical difference in the host immune response against distinct Ad vectors, a situation that has implications for gene delivery and vaccine development.