Yellow fever (YF) vaccines are highly efficacious in preventing mortality and morbidity in humans living in or traveling to regions of tropical Africa and South America where YF is endemic (3,20). To date, more than 400 million doses of YF vaccines have been administered (20). Currently licensed YF vaccines include the attenuated 17D strain, which is propagated in chicken embryos. Vaccine preparation typically includes inoculation of 7-to 9-day-old embryonated eggs with the vaccine strain. Infected embryos are harvested, pooled, and then homogenized. Homogenates are clarified by low-speed centrifugation, and the supernatant fluid containing the vaccine harvest is titrated and lyophilized under standardized conditions (33).Because of the risk of contamination with substrate-derived avian pathogens, regulations for vaccine manufacture require all avian embryos used for propagation of vaccine virus to be derived from a closed, specific pathogen-free (SPF) flock devoid of known avian pathogens including exogenous retroviruses of the avian leukosis virus (ALV) and the reticuloendotheliosis virus groups (32). Despite the use of SPF chicken flocks, low-level reverse transcriptase (RT) activity, an indication of retroviruses, was recently detected in YF vaccines and other chick cell-derived measles and mumps vaccines produced by several manufacturers in Europe and the United States (5,19,28). The origin of RT activity has thus far been examined in measles vaccines produced in chicken embryonic fibroblasts (CEF). These studies have shown that this RT activity was associated with particles containing RNA from endogenous avian virus (EAV) and endogenous ALV (ALV-E) (17,28,30). These findings were consistent with current vaccine manufacturing regulations that require the elimination of exogenous retroviral infections from source chickens. These manufacturing regulations do not address the presence of endogenous retroviruses because such particles have not previously been known to be associated with chick cell-derived vaccines.Both EAV and ALV-E are members of endogenous retroviral families present in the chicken germ line. Little is known about the EAV family, which is distinct from but related to the ALV family. EAV elements are present in at least 50 copies per chicken genome (23). The majority of EAV loci remain uncharacterized, and the nucleotide sequences of full-length EAV genomes are not available. Partial EAV sequences show numerous frameshift mutations or sizable deletions in the env region (6).