Technical Abstract: Beginning in April 2009, cases of acute respiratory disease were reported in humans caused by a novel H1N1 influenza A virus (pH1N1) in Mexico which has since spread globally in the human population and been declared a pandemic. Initial studies using intranasal route of inoculation failed to produce infection in turkeys, chickens and ducks, suggesting the pandemic H1N1 virus had low potential for natural respiratory infections in poultry. However, isolated cases of natural infections with drops in egg production have been reported in turkey breeder hens in Chile, Canada and the USA suggesting a reproductive and not a respiratory route of infection was more plausible. To understand the role of route of inoculation on infectivity and pathogenesis, reproductively active turkey breeder hens were challenged by intranasal (IN), intracloacal (IC) or intrauterine (IU) inoculation with a human Chilean pH1N1 virus. No clinical disease was produced with IN inoculation, but IU and IC inoculation produced transient diarrhea. By 5 days after challenge, the IU turkeys stopped laying eggs, but IC inoculated turkeys laid eggs daily until 9 days and the IN inoculated turkeys laid eggs daily until termination of the study. These data suggest turkeys hens can be infected by inoculation via the reproductive tract or lower digestive tract which could occur in the field through the human-controlled activity of artificial insemination of turkey breeder hens.