The extent to which rats express anxiety-like behavior on the elevated plus-maze (EPM) depends on their previous maze experience. Open-arm avoidance develops in maze-experienced rats, and is often accompanied by a diminished anxiolytic response to benzodiazepines. Regions of the dorsal raphe nucleus (DRN) were examined in male Sprague-Dawley rats using c-Fos and serotonin immunohistochemistry following a single exposure, a second exposure or no exposure to the EPM. We then examined the effect of the benzodiazepine anxiolytic chlordiazepoxide (CDP, 5 mg/kg) on EPM behavior and DRN neural activity. Enhanced open-arm avoidance was evident on the second EPM trial in both experiments. The observed pattern of c-Fos expression suggests that the first exposure to the maze activates serotonin cells in the rostral and dorsal regions of the DRN and that only the dorsal subregion is activated by a second exposure. CDP increased open-arm exploration during the first trial, which corresponded to decreased 5-hydroxytryptamine (5-HT) activity in the rostral and ventral subregions of the DRN. However, 5-HT activity in the DRN was reduced in rats on the second maze trial compared with the first trial, when CDP had no effect on open-arm exploration. These results suggest that open-arm avoidance in maze-experienced rats can be characterized as a coping response that is mediated by specific populations of 5-HT neurons in the DRN.