BACKGROUND: Bedside assessments of cerebral oxygenation are sought to monitor cerebral injury in patients undergoing intensive care. Spatially resolved spectroscopy measures tissue oxygenation index (TOI, ) which reflects mixed cerebral arterial and venous oxygenations. We aimed to evaluate arterial and venous components of TOI (cerebral arterial to venous volume ratio [A:V ratio]) in the newborn lamb brain using cerebral arterial and venous blood samples, and to investigate the impact of acute hypoxemia on the A:V ratio and TOI. METHOD: Nine lambs were ventilated with varied inspired oxygen to generate arterial oxygen saturations between 25 and 100 . Cerebral arterial and venous oxygen saturations analyzed using cooximeter of arterial and superior sagittal sinus blood were used to estimate TOI (TOIcox), assuming cerebral A:V ratio of 25:75. TOIcox was compared with the TOI measured by spatially resolved spectroscopy (TOIsrs). Actual cerebral arterial and venous volume fractions were reestimated using TOIsrs = cerebral arterial volume fraction cerebral arterial oxygen saturation + cerebral venous volume fraction*cerebral venous oxygen saturation. RESULTS: Median (range) TOIsrs was 48.5 (32.0-64.1 ), and TOIcox was 48.4 (13.7-74.4 ), and the two were significantly correlated (R = 0.77). The mean difference between TOIsrs and TOIcox was 2.4 (limits of agreement +/- 18.1 ). The TOIsrs - TOIcox difference varied with oxygen saturations, with TOIsrs higher than TOIcox at low saturations, and lower at high saturations. Cerebral arterial volume fraction was 22.9-27.5 in normoxia and markedly increased in hypoxemia. CONCLUSION: TOI corresponds with cerebral oxygenation. The variable agreement of TOIsrs with TOIcox may reflect changes in cerebral A:V ratio due to arterial oxygenation-related vasoreactivity.