A large nesting colony of Ardeid birds at the Finney-Ramer Wildlife Refuge in Imperial County, California, did not appear to be a focus of West Nile virus (WNV) amplification during the summer of 2004. Blood samples taken during June and July from 155 nestlings of four species of Ardeid birds (cattle egrets, black-crowned night herons, great egrets, and snowy egrets) and five nestling double-crested cormorants yielded a single WNV isolation from a 3-week-old cattle egret. Antibody was detected by enzyme immunoassay from 20 nestlings (13%), 14 (70%) of which were confirmed as positive by plaque reduction neutralization test (PRNT). However, titration end points against WNV and St. Louis encephalitis virus (SLEV) were similar precluding viral identification. The grouping of positives within few nests, highest PRNT titers in youngest birds (<1 weeks of age), the decline of titer with nestling age, and the lack of antibody specificity indicated that antibody may have been acquired maternally and did not represent new infections. Infection rates in Culex tarsalis mosquitoes collected near the Ardeid colony at Ramer Lake (3.1 per 1,000) were statistically similar to rates estimated at the nearby Wister Unit wetlands (5.3 per 1,000) that lacked an Ardeid nesting colony. Black-crowned night heron nestlings experimentally infected with the NY99 strain of WNV produced viremias >5 log10 plaque forming units (PFU)/mL and were considered moderately competent hosts, whereas cattle egret nestlings had viremias that remained <5 log10 PFU/mL and were incompetent hosts.