An environmental preference test chamber (EPTC) was designed, constructed, and utilized in an initial test for response of laying hens to atmospheric ammonia. The EPTC featured four interconnected, individually ventilated clear acrylic compartments. Each compartment contained a wire-mesh cage that was divided into two sections, one section used for a test bird to navigate between the compartments and the other section used for three stimulus birds to reside in each compartment. The EPTC was designed to assess individual bird preference without isolation effects. The section dividers may be removed to assess group preference. An initial experiment was conducted with six test hens to assess bird aversion to atmospheric ammonia. Each hen was trained to navigate the inter-compartment door prior to the aversion test. Following one day of acclimation to the chamber, behavioral data of the hen were collected for 2 days at ambient conditions (baseline) and 3 days at ammonia level of 25 ppm vs. <10 ppm. Hen location (compartment) was documented and compared for baseline and treatment periods. All hens learned to navigate the chamber within 10 h; 4 of the 6 hens learned within 2 h. No preference for the level of ammoniated condition was observed with regard to occupancy of the corresponding compartment or number of entries into each environment. Further investigation is warranted to determine if this finding is a lack of aversion or other phenomenon. The EPTC will also enable future users to examine preference responses of hens to other environmental conditions, such as thermal comfort vs. air quality.