This study related the replication of an H9N2 avian influenza virus in chickens to the induction of host acute immune response after aerosol or intranasal inoculation with the virus. On 1, 2, 4, and 7 days postinoculation (dpi), oropharyngeal swabs and tissue specimens of trachea, lungs, spleen, and cecal tonsils were collected for quantification of viral RNA. Expression of cytokine genes in lungs, spleen, and cecal tonsils was quantified by reverse transcriptase-PCR. Virus was detected in all oropharyngeal swabs up to 4 dpi in chickens from both inoculation groups. However, virus was detected more frequently (P < 0.05) and in higher titers (1–4 log difference) in specimens of trachea and lungs from the group exposed to aerosols than from the group given intranasal drops. In accord with viral replication findings, expressions of cytokine genes interleukin (IL)-1β (on 2 and 7 dpi), IL-6 (on 2 dpi), and interferon (IFN)-γ (on 2 and 4 dpi) were up-regulated to a significantly higher level (P < 0.05) in lung tissue specimens from the group exposed to virus aerosol than from controls that were given saline intranasally. Only IFN-γ on 1 dpi was up-regulated (P < 0.05) above that of controls in lung tissue specimens from the group given intranasal drops of virus. In comparison, replication of the virus and induction of IL-1β and IL-6 genes were limited in spleen and cecal tonsil tissue specimens from both groups of chickens inoculated with the virus. These findings indicate that virus administered in aerosols was more efficient than virus administered as intranasal drops, in infecting the lower respiratory tract and in inducing the activity of the cytokine genes. The intense respiratory infection caused by virus aerosols might increase the shedding and transmission of the H9N2 virus in chickens.