Background Avian influenza (AI) infection in poultry can result in high morbidity and mortality, and negatively affect international trade. Because most AI vaccines used for poultry are inactivated, our knowledge of immunity against AI is based largely on humoral immune responses. In fact, little is known about cellular immunity following a primary AI infection in poultry, especially regarding cytotoxic T lymphocytes (CTL’s). Methods In these studies, major histocompatibility complex (MHC)-defined (B2/B2) chickens were infected with low pathogenic AI (LPAI) H9N2 and clinical signs of disease were monitored over a two weeks period. Splenic lymphocytes from infected and na ve birds were examined for cross reactivity against homologous and heterologous (H7N2) LPAI by ex vivo stimulation. Cellular immunity was determined by cytotoxic lysis of B2/B2 infected lung target cells and proliferation of T cells following exposure to LPAI. Results Infection with H9N2 resulted in statistically significant weight loss compared to sham-infected birds. Splenic lymphocytes derived from H9N2-infected birds displayed lysis of both homologous (H9N2) and heterologous (H7N2) infected target cells, whereas lymphocytes obtained from sham-infected birds did not. T cell proliferation was determined to be highest when exposed to the homologous virus. Conclusions Taken together these data extend the findings that cellular immunity, including CTL’s, is cross reactive against heterologous isolates of AI and contribute to protection following infection.