Avian influenza virus (AIV) was studied in ring-billed gulls (Larus delawarensis) in one breeding colony on Lake Erie in 2000, and two on Lake Ontario in both 2000 and 2004. Antibodies to H13 AIV were detected in 92% of adults in 2000 and 82% in 2004. Antibody prevalence in 3-wk-old chicks was 5%–30% (overall 15%) in 2000 and 21% and 76% (overall 48%) in 2004. In 5-wk-old chicks, antibody prevalence was 23%–75% (overall 53%) in 2000 and 53% and 79% (overall 66%) in 2004. Geometric mean antibody titers at 3 and 5 wk did not differ in 2000, but increased significantly at one colony in 2004. In 2000, overall prevalence of AIV isolation from cloaca in embryonated chicken eggs was 32% (3 wk old), 13% (5 wk old), and 0 (adults), but AIV was also isolated from kidney and lung in a high proportion of tissues cultured from 3-wk-old birds in one colony. Isolates from cloaca were characterized as subtype H13 by serology; all 15 tested for neuraminidase were H13N6. However, three AIV detections considered on the basis of nucleotide sequence to be subtype H16 were among the 28 detected retrospectively by PCR in archived cloacal swabs; the remainder were subtype H13. Outcome of virus isolation was not related to presence of antibody titers in chicks. The presence of antibody to AIV in chicks was associated significantly with inflammation in heart, kidney, pancreas, and liver. AIV was not isolated in 2004. AIV infected chicks annually within the first 3 wk of life, ultimately infecting the majority of birds in most colonies, but did not appear to cause clinical disease.