The protective effect of the A/Ck/Yoko/aq55/01 (H9N2) avian influenza virus against the highly pathogenic H5N1 virus, i.e., A/Ck/Yama/7/04 (genotype V), was examined. Three 5-week-old chickens were inoculated intranasally with the H9N2 virus (10(8.6) EID(50)/head) and were kept with two contact chickens. All of the infected chickens were reinoculated with the same virus at 20 weeks of age, and 10 days later, they were challenged intranasally with the H5N1 virus (10(4.0) EID(50)/head). Five chickens simultaneously challenged with only the H5N1 virus (challenge control) died within 4 days postchallenge (d.p.c.). In contrast, four out of the five challenged, immune chickens died from 5 to 8 d.p.c. The median time to death in the immune chickens (6.3 days) was significantly longer than that in the challenge controls (3.4 days) (P < 0.01). No H5N1 virus shedding into the tracheae and feces of the challenged, immune chickens were detected for 3 d.p.c., but H5 genes were detectable in only one chicken by a loop-mediated isothermal amplification method. The H5N1 viruses were detected in the tracheae and/or feces of the dead immune chickens at death or 1 to 2 days before death. Only one out of the five challenged, immune chickens survived the H5N1 challenge without any signs for 14 d.p.c., but the virus and H5 gene were sporadically detected in the trachea only 7 and 14 d.p.c., respectively. This study shows that the H9N2 viruses may have the potential to induce cross-protection to the challenge with a recent lethal H5N1 virus (genotype V).