The avian immune system has been shown to possess a repertoire of cytokines directing T-helper (Th) 1 and Th2 types of immune responses similar to that in mammals. The objective of this study was to establish in situ hybridization (ISH) for the localization of mRNA of selected signal cytokines, chicken interferon-γ (ChIFN-γ), chicken interleukin (ChIL)-4 and ChIL-13 in fixed tissues. RNA probes were generated to hybridize to 488, 318, and 417bp of the respective target mRNA. Probe concentrations ranging from 100ng/ml to 400ng/ml were shown to be suitable to label cells that expressed these cytokines. The specificity of every probe was verified using the respective sense probe. ChIFN-γ, ChIL-4 and ChIL-13 positive cells were observed in the lymphocytic infiltrations of liver and in the periarteriolar lymphatic sheaths of spleen collected from specific-pathogen-free chickens. ISH of these cytokines in a severely inflamed liver due to infiltration with the parasite Histomonas meleagridis revealed the expression of both ChIFN-γ and ChIL-13 mRNA in the mononuclear infiltrates. In conclusion, ChIFN-γ, ChIL-4 and ChIL-13 mRNA were efficiently localized by ISH, which supplies a valid technique to characterize immune responses in fixed tissues.