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 x 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 1(33)xenon method.