Both acute and chronic tolerance can develop to allopregnanolone-a gamma-aminobutyric acid (GABA)-modulatory progesterone metabolite. Here we investigated if acute tolerance to allopregnanolone persisted for 1 or 2 days after the induction and thus could be the initial part of chronic tolerance. Male rats were anaesthetised with allopregnanolone (i.v) to the deep anaesthesia level of the silent second (SS), which is an EEG burst suppression of 1 s or more. They were divided into four groups: SS1-anaesthesia to the first silent second; LAn (long anaesthesia)-90 min anaesthesia at the SS level; SS2;D1-90 min anaesthesia and SS induction 1 day later; SS2;D2-90 min anaesthesia and SS induction 2 days later. Allopregnanolone concentrations in tissue and serum were analysed. Levels of the GABAA receptor alpha2, alpha4, gamma2(S+L) and delta subunits mRNAs were analysed by in situ hybridisation. Acute tolerance was induced during the 90 min anaesthesia. Tolerance persisted for 1 day, since the dose of allopregnanolone needed to induce a new SS anaesthesia was increased after 1 day. The level of alpha4 subunit mRNA expression in the ventral posteriomedial nucleus of thalamus was negatively related to the tolerance parameters, the SS dose of allopregnanolone and DeltaSS (SS dose difference between days). Allopregnanolone threshold anaesthesia lasting 90 min induces acute tolerance that persisted for at least 1 day, which could be used as the start of a chronic tolerance. The alpha4 subunit may be involved in allopregnanolone caused effects in the brain.