Alfaxalone (3α-hydroxy-5α-pregnane-11, 20-dione) is a neuroactive steroid with anaesthetic properties and a wide margin of safety. The pharmacokinetic properties of alfaxalone administered intravenously and intraperitoneally in rats (n = 28) were investigated. Mean t1/2elim for 2 and 5 mg/kg i.v. was 16.2 and 17.6 min, respectively, but could not be estimated for IP dosing, due to sustained plasma levels for up to 60 min after injection. Clp for i.v. injection was calculated at 57.8 ± 23.6 and 54.3 ± 6.8 mL/min/kg, which were 24.5% and 23% of cardiac output, respectively. The observed Cmax was 3.0 mg/L for IP administration, and 2.2 ± 0.9 and 5.2 ± 1.3 mg/L for 2 and 5 mg/kg i.v. administration, respectively. AUC0–60 was 96.2 min·mg/L for IP dosing. The relative bioavailability for IP dosing was 26% and 28% compared to i.v. dosing. Differences in t1/2elim and Clp from previous pharmacokinetic studies in rats are likely due to variations in alfaxalone formulation rather than sex differences. Alfaxan® given IP caused sustained levels of alfaxalone, no apnoea and longer sleep times than i.v. dosing, although immobilization was not induced in 30% of rats given Alfaxan® IP. A pharmacodynamic study of the effects of combining IP injection of Alfaxan® with other premedication agents is worthwhile, to determine whether improved anaesthesia induction could ultimately provide an alternative anaesthetic regimen for rats.