We have recently shown that live attenuated virus vaccines produced on chicken-derived cells contain low levels of particle-associated reverse transcriptase (RT). In both virus and corresponding control harvests produced on chicken embryo fibroblasts, these activities were present at significantly higher concentrations than in the vaccines. In order to identify the putative retrovirus sequence responsible for this activity, a novel method for the selective PCR amplification of particle-associated retrovirus RNA that uses DNA primers complementary to the primer binding sites of the known exogenous retroviruses in combination with an anchor primer was applied. A product of the endogenous avian retrovirus family EAV-0, termed EAV-0(B1), was reproducibly generated with a tRNA(Trp)-derived primer from the RT peak fraction of a sucrose density gradient run with a harvest of a live attenuated measles vaccine. In contrast, no products were detected with primers derived from tRNA(Pro), tRNA(Lys)1,2 or tRNA(Lys)3. In the same fraction, genomic RNA of EAV-0(B1) was demonstrated by long PCR. Analysis of several sucrose density gradients from different harvests of various manufacturers demonstrated accumulation of, and colocalization with, RT activity for the EAV-0(B1) RNA but not for a chicken cellular mRNA. Synthesis of cDNA from EAV-0(B1) RNA was shown by endogenous RT reaction. Furthermore, complexes of naturally primed EAV-0(B1) RNA with RT were demonstrated. Taken together, these data strongly suggest that EAV-0 is able to produce virus-like particles with an active RT.