Technical Abstract: Gulls are widely recognized reservoirs for low pathogenic avian influenza (LPAI) viruses; however, the subtypes maintained in these populations and/or the transmission mechanisms involved are poorly understood. Although, a wide diversity of influenza viruses have been isolated from gulls, two hemagglutinin subtypes (H13 and H16) are rarely detected in other avian groups and existing surveillance data suggests they are maintained almost exclusively within gull populations. In order to evaluate the host range of these gull-adapted influenza strains and to characterize viral infection in the gull host, we conducted a series of challenge experiments, with multiple strains of H13 LPAI, in juvenile ring-billed gulls (Larus delawarensis; one strain), mallards (Anas platryrhyncos; three strains), chickens (Gallus domesticus; eight strains), and turkeys (Meleagris gallopavo; eight strains). The susceptibility to H13 LPAI viruses varied between species and viral strain. Gulls were highly susceptible to H13 LPAI virus infection and excreted virus via the oropharynx and cloaca for several days. The quantity and duration of shedding was similar between the two routes. Turkeys and ducks were resistant to infection with multiple isolates of H13 LPAI, but low numbers of inoculated birds were infected after challenge with specific strains. Chickens were refractory to infection with all isolates of H13 LPAI virus. The results of these challenge studies are consistent with existing surveillance and experimental data on H13 LPAI viruses. Collectively, these data suggest that influenza viruses of the H13 subtype are strongly host-adapted to gulls, but can rarely spill-over and infect other groups of birds.