In order to develop better control measures against avian influenza, it is necessary to understand how the virus transmits in poultry. In a previous study in which the infectivity and transmissibility of the pandemic H1N1 influenza virus was examined in different poultry species, we found that no or minimal infection occurred in chicken and turkeys intranasally (IN) inoculated with the virus. However, we demonstrated that the virus can infect laying turkey hens by the intracloacal (IC) and intraoviduct (IO) routes, possibly explaining the drops in egg production observed in turkey breeder farms affected by the virus. Such novel routes of exposure have not been previously examined in chickens and could also explain outbreaks of low pathogenicity avian influenza (LPAI) that cause a decrease in egg production in chicken layers and breeders. In the present study, 46-wk-old specific-pathogen-free chicken layers were infected by the IN, IC, or IO routes with one of two LPAI viruses: a poultry origin virus, A/chicken/CA/1255/02 (H6N2), and a live bird market isolate, A/chicken/NJ/12220/97 (H9N2). Only hens IN inoculated with the H6N2 virus presented mild clinical signs consisting of depression and anorexia. However, a decrease in number of eggs laid was observed in all virus-inoculated groups when compared to control hens. Evidence of infection was found in all chickens inoculated with the H6N2 virus by any of the three routes and the virus transmitted to contact hens. On the other hand, only one or two hens from each of the groups inoculated with the H9N2 virus shed detectable levels of virus, or seroconverted and did not transmit the virus to contacts, regardless of the route of inoculation. In conclusion, LPAI viruses can also infect chickens through other routes besides the IN route, which is considered the natural route of exposure. However, as seen with the H9N2 virus, the infectivity of the virus did not increase when given by these alternate routes.