Urethane is often used as a sole anaesthetic agent for non-recovery studies in laboratory animals. However, the use of urethane is controversial, in part, because the electroencephalogram after urethane administration is similar to the electroencephalogram recorded from unanaesthetized animals. Here, we assessed the minimum alveolar concentration (MAC)-sparing effects of urethane by measuring the effect of two doses of urethane on the MAC of isoflurane in male Sprague Dawley rats. Isoflurane MAC was measured before and after intravenous administration of urethane at 1.0 g/kg (Group G₁, n = 6) and 1.5 g/kg (Group G₁.₅, n = 6), or an equal volume of 0.9% saline (Group Gs, n = 6). Baseline isoflurane MAC was not statistically different between groups (isoflurane concentration: 1.47 ± 0.08%, 1.40 ± 0.19% and 1.42 ± 0.12% for G₁, G₁.₅ and Gs, respectively). Intravenous injection of saline did not alter isoflurane MAC (post-saline MAC: 1.43 ± 0.11%). After urethane administration, isoflurane MAC decreased in a dose-dependent manner (new MAC G₁: 0.19 ± 0.06%; G₁.₅: 0.03 ± 0.01%; P < 0.05). The isoflurane MAC after 1.5 g/kg urethane was not significantly different from room air isoflurane concentrations (0.01 ± 0.01%), demonstrating a 100% MAC reduction at this dose. In conclusion, high-dose urethane (1.5 g/kg intravenously) was suitable as a sole anaesthetic agent to prevent gross purposeful movement during the conditions of the study, whereas low-dose urethane (1.0 g/kg intravenously) was not.