Anxiolytics specifically increase the number of exploratory transitions in a two-chambered model system for anxiety in mice. Characterization of parameters to optimize and standardize this model required analysis of multiple use of test animals, intertrial interval, and circadian variability. Time of day did not affect exploratory activity in mice treated with vehicle or diazepam between 10 a.m. and 11 p.m. Intertrial intervals of 1, 3, 5, or 7 days were equally effective. The diazepam-induced increase in exploratory activity was significant over the first three uses of test animals. These data recommend reuse of mice to a maximum of three trials, throughout the daytime or evening hours of their lighting schedule.