Abolishment of anxiolytic-like effects of diazepam occurs during re-exposure to some animal tests of anxiety. We investigated the loss of anxiolytic-like effects of diazepam during Trial 2 on previously undrugged mice, namely one-trial tolerance (OTT). Swiss mice were subjected to 1) Four-Plate Test (FPT) without punishments in Trial 1 or 2) FPT without punishments in both Trials or 3) FPT with spatial modifications in Trial 1 or 4) Elevated Plus Maze (EPM), then 24 h later to FPT, with saline, diazepam (1 mg/kg) or DOI (1 mg/kg). Removing punishments in Trial 1 does not counteract the effect reduction of diazepam in Trial 2, but spatial modifications of the aversive environment. Previous exposure to EPM does not trigger a loss of efficacy of diazepam in FPT. Electric punishments do not trigger OTT to benzodiazepines; whilst knowledge of the environment seems to be responsible for this phenomenon. FPT may be useful to study OTT because punishments potentate OTT in this model of anxiety.