Numerous methodological-related variables have been demonstrated to influence the baseline anxiety level of rodents exposed to the elevated plus-maze (EPM), raising questions about the sensitivity of this test for the detection of the effects of anxiolytic drugs. Thus, the present study was designed (1) to assess the combined effects of illumination (40-lx red or white light), closed wall type (walls made of translucent or opaque material) and extramaze space size (small or spacious experimental room) on rat behaviour, and (2) to investigate the effects of such parameters on the relevance of the maze for detecting the effects of diazepam orally administrated at the anxiolytic dose of 3 mg/kg. Results indicate that illumination and closed wall type are two main independent parameters that are able to modify the open arm avoidance. Moreover, the closed wall type interacts with the extramaze space size since the reduction of the open arm exploration induced by opaque closed walls is two-fold stronger in the spacious experimental room than in the small one. Finally, the diazepam anxiolytic activity is significantly detected in our laboratory in specific EPM conditions (maze with opaque walls, use of a red light, maze located in a spacious experimental room). In conclusion, the present study demonstrates that an inappropriate baseline anxiety level due to the methodological use of the EPM can dramatically reduce the sensitivity of the maze for the detection of benzodiazepine-related compounds. This study also provides new insights into the perception of the EPM open space in rats.