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Ammonia (NH3) is a potential health hazard to both humans and animals, causing local and systemic low-grade inflammation based on its levels and exposure durations. The objective of this study was to examine the effects of 45 wk of exposure to 30 ppm NH3 on the concentrations of acute-phase proteins, immunoglobulins, and cytokines in laying hens. At 18 wk of age, a group of Hy-Line W-36 hens was randomly assigned to 4-hen cages. These cages were evenly divided between 2 environmentally controlled chambers. At 25 wk of age, one chamber was maintained continuously with fresh air (NH3 < 5 ppm; control group) and the other one was injected with NH3 and controlled at 30 ppm (NH3 group) for 45 wk. At 70 wk of age, blood and spleen samples (n = 8 per treatment) were collected for analyses of immunological parameters. No significant differences were observed in plasma levels of albumin, complement components (C)-3 and C-4, immunoglobulin (Ig)M, IgA and IgG, interleukin (IL)-6, IL-10, or interferon gamma in the NH3 group compared to control group. Compared to control hens, NH3 exposed hens had higher plasma levels of α-1-acid glycoprotein (24%) and tumor necrosis factor alpha (54%) and higher mRNA expression of IL-1β (47%) and IL-6 (62.5%) in the spleen. These results indicated that hens may have the capability to adapt to chronic effects of moderate levels of NH3. Future studies should explore acute effects of NH3 at higher levels on hen health and welfare.