The specific-pathogen-free (SPF) flocks of chickens maintained by the Department of Microbiology and Immunology at Cornell University became infected, inadvertently, with chicken anaemia virus (CAV), as demonstrated by seroconversion. Chickens from five flocks representing three different strains were examined for the presence of CAV using nested PCR. Virus was detected in ovaries, infundibula, vas deferentia, testes and spleens. Ovaries were positive in 38 to 72% of the hens in four flocks with 13 to 56 birds examined per flock. Interestingly, the ovaries were often the only positive tissues, while a few hens had only positive spleens. In roosters, the vas deferens was positive in 30 to 79% of the birds with 5 to 19 birds examined per flock; the vas deferens was the only positive tissue in 20 to 37%. Individual cells in the theca externa and rare epithelial cells in the infundibular epithelium were positive tissue in 20 to 37%. Individual cells in the theca externa and rare epithelial cells in the infundibular epithelium were positive for CAV by in situ PCR. Positive cells were not detected in testes or vas deferentia. The SH-1 strain of CAV was isolated from these tissues and partially sequenced. Only minor sequence differences were found compared to CIA-1 and Cux-1. Embryos from matings between persistently infected dams and sire had CAV-positive cells in mesenchyme near the developing vertebral column. The data show that CAV persists in the reproductive tissues far longer than previously thought, and that it can be vertically transmitted from persistently infected birds.