A novel environmental preference chamber (EPC) was developed and used to assess responses of laboratory mice to atmospheric ammonia. The EPC features 1) a test chamber with 4 individually ventilated, mutually accessible compartments; b) automated tracking of mouse movements by using paired infrared sensors; c) identification of individual mice by using photosensors; d) monitoring and regulation of the NH3 concentration in each compartment; and e) personal-computer-based data acquisition. In an initial preference study with the EPC, 4 groups of 4 laboratory mice (BALB/c/Bkl; body weight, 13.4 to 18.4 g) were each given a choice among 4 NH3 concentrations (mean +/- SE) of 4 +/- 2, 30 +/- 2, 56 +/- 4, and 110 +/- 6 ppm for 2 d after a 2-d familiarization period. Once trained to use the intercompartment tunnels, the mice made extensive use of the EPC, with each group making more than 2000 intercompartment movements during 48 h. Video recording verified the results of the automatic tracking system, which detected and correctly determined mouse location for 79% of the moves. The use of photosensors proved to be ineffective in recognizing individual mice. Although the EPC would benefit from refinement and further development, it simplified analysis of locomotion behavioral data. Results of the preference study indicated that the mice exhibited no clear preference for, or aversion to, any of the experimental concentrations of ammonia and that the mice clearly preferred the upper 2 compartments of the chamber over the lower 2 compartments. Further investigation should be conducted to verify these preliminary results and explore other preferences of laboratory mice for environmental conditions and resources.