Culex pipiens form pipiens and Cx. pipiens form molestus (Diptera: Culicidae) belong to a cosmopolitan taxonomic group known as the Pipiens Assemblage. Hybridization between these forms is thought to contribute to human transmission of West Nile virus (WNV) in North America. Complementary choice and no-choice landing assays were developed to examine host acceptance by North American Cx. pipiens in the laboratory. Populations collected from above- and below-ground sites in suburban Chicago were identified as forms pipiens and molestus using a polymerase chain reaction-based assay. Avian and human host acceptance was then quantified for the two populations, as well as for their hybrid and backcross offspring. No-choice tests were used to demonstrate that both the pipiens and molestus forms were capable of feeding on human and avian hosts. Choice tests were used to demonstrate that form pipiens females were strongly avian-seeking; an individual's probability of accepting the chick host was 85%. Form molestus females were more likely to accept the human host (87%). Rates of host acceptance by F1 and backcross progeny were intermediate to those of their parents. The results suggest that host preferences in Cx. pipiens are genetically determined, and that ongoing hybridization between above- and below-ground populations is an important contributor to epizootic transmission of WNV in North America.