The infectivity, transmission, and pathogenicity potential of avian influenza virus (AIV) subtype H16N3, isolated from the European herring gull (Larus argentatus), was examined in chickens. Nineteen 6-wk-old commercial Lohmann white chickens were inoculated intranasally with 1 × 106 50% egg infectious dose and clinical signs, humoral immune response, virus shedding, virus transmission, and pathologic changes in the respiratory tract were studied. Oropharyngeal and cloacal swabs were collected for viral RNA detection by real-time reverse transcriptase–PCR (rRT-PCR). Sera were collected and examined for H16-specific antibodies using a hemagglutination inhibition test. Tissue samples from the nasal cavity, trachea, and lung were collected at postmortem examination for histopathology and viral RNA detection by rRT-PCR. In one bird, bilateral serous nasal discharge was observed at 2 days postinoculation (DPI) and viral RNA was detected in oropharyngeal swabs at 2 and 4 DPI. Viral RNA was also detected from the oropharynx of an additional bird at 5 DPI. Moreover, H16-specific antibodies were detected in sera from these two birds at 14 and 21 DPI. No viral RNA was detected from cloacal swabs, and no virus transmission between virus-inoculated chickens and noninoculated contact chickens was observed. Tissue samples from the nasal cavity, trachea and lung were negative for viral RNA and no gross or histopathologic lesions were observed in the virus-inoculated birds. These results indicate that gull-derived AIV subtype H16N3 causes only limited infection in chickens under experimental conditions.