Laser Doppler flowmetry (LDF) is a recent technique that is increasingly being used to monitor relative changes in cerebral blood flow whereas the intra-arterial ¹³³xenon injection technique is a well-established method for repeated absolute measurements of cerebral blood flow. The aim of this study was to validate LDF for assessment of cerebral autoregulation and CO₂ reactivity with the ¹³³xenon injection technique as the gold standard. Simultaneous measurements of cerebral blood flow (CBF) were collected by LDF (CBF_LDF) and the ¹³³xenon method (CBF_Xe) while (1) cerebral autoregulation was challenged by controlled systemic haemorrhage, or (2) cerebral blood flow was varied by manipulating the arterial partial pressure of CO₂ (P_a,CO2). LDF slightly overestimated CBF under conditions of haemorrhagic shock and haemodilution caused by controlled haemorrhage (paired t test, P < 0.05). However for pooled data, the autoregulation lower limit was similar when determined with the ¹³³xenon and the LDF techniques: 65 ± 3.9 mmHg and 60 ± 5.6 mmHg, respectively. Linear regression analysis yielded CBF_Xe= (1.02 × CBF_LDF) + 9.1 and r= 0.90. Even for substantial changes in P_a,CO2, the two methods resulted in similar results. We conclude that even though LDF overestimated CBF during haemorrhagic shock caused by controlled haemorrhage, the lower limit autoregulation was correctly identified. The laser Doppler technique provides a reliable method for detection of a wide range of cerebral blood flow changes under CO₂ challenge. Haemodilution influences the two methods differently causing relative overestimation of blood flow by the laser Doppler technique compared to the 1³³xenon method.