The quantitative real time reverse polymerase chain reaction (RRT-PCR) is to date the preferred test method for the diagnosis of avian influenza (AI), which can be performed only in specialized laboratories. Different antigen detection methods for the diagnosis of AI were previously reported to be specific and sensitive in field outbreaks. These tests can be performed in simple country site labs. Brain smears of domestic birds (n = 105) collected during AI field outbreaks were examined with immunocytochemistry (IC). The results were statistically analyzed by comparing IC to brain histology (BH), and immunohistochemistry (IHC), to gross pathological examination (GP) (n = 105) and to RRT-PCR (n = 91). AI was diagnosed with RRT-PCR in 66 cases. IC and IHC were positive in 59/66 (90 %) and 60/66 (91 %) cases respectively. Lesions suspicious for AI were detected with GP and HP in 66/66 (100 %) and 61/66 (92 %) cases respectively. An almost perfect agreement was found between RRT-PCR, IC, IHC, and HP. Substantial agreement was found between IC and GP, between IHC and GP, between HP and GP, and between RRT-PCR and GP. The chromogen-based IC test presented in this study is allowed to produce a durable staining, which can be evaluated using a simple brightfield microscope. The test is rapid (can be completed in 2 hours), sensitive (90 %), specific (100 %), and cost-effective, which made the method suitable for routine diagnostic test in AI epidemics.