Laying hens are chronically exposed to high levels of ammonia (NH3), one of the most abundant aerial pollutants in poultry houses. Tests for aversion to NH3 in laying hens have used artificially sourced NH3/air mixtures (i.e., from a gas cylinder) showing that birds prefer fresh air to NH3. However, artificially sourced NH3/air mixtures may not accurately reflect barn air conditions, where manure emits a variety of gases. Herein, we investigated whether laying hens differentiate between artificially and naturally sourced NH3/air mixtures and how exposure to NH3 affects foraging and aversive behavior. A total of 20 laying hens was exposed to artificially sourced [A] (from an anhydrous NH3 cylinder) and naturally sourced [N] (from conspecific laying hen excreta) gas mixtures. Hens were exposed to A and N mixtures with NH3 concentrations of 25 and 45 ppm, as well as fresh air [FA]. During the experiment, all birds were exposed to each treatment 3 times using a custom-built polycarbonate chamber, containing a foraging area (containing raisins, mealworms, and feed mix) and a gas delivery system. All testing sessions were video recorded, analyzed with INTERACT® software, and subjected to a GLIMMIX procedure in SAS. Our results showed that the laying hens spent less time foraging overall (P < 0.001) and were slower to commence foraging (P = 0.004) in ammoniated environments compared to the fresh air. Laying hens were more likely to forage for a longer time (with fewer interruptions) in N than in A treatments (P < 0.001). Laying hens also reacted with greater aversion towards treatment A compared to treatment N (P < 0.001). These findings suggest that the laying hens of our study preferred fresh to ammoniated air and that they behaved differently in artificially and naturally sourced NH3/air mixtures, possibly due to the presence of familiar stimuli from the excreta. These findings have implications for new developments in methodological approaches for behavioral testing and for recommendations regarding NH3 levels inside poultry barns. © 2017 Poultry Science Association Inc.