The relative magnitude of vascular reactivity to inhaled gas stimuli in the major retinal arterioles has not been systematically investigated. The purpose of this study was to compare the magnitude of retinal vascular reactivity in response to inhaled gas provocation at equivalent measurement sites in the superior-, and inferior-, temporal retinal arterioles (STA, ITA). One randomly selected eye of each of 17 healthy volunteers (age 24.4 ± 4.7) was prospectively enrolled. Volunteers were connected to a sequential gas delivery circuit and a computer-controlled gas blender (RespirActâ„¢, Thornhill Research Inc., Canada) and underwent an isocapnic hyperoxic challenge i.e. P(ET)O(2) of 500 mm Hg with P(ET)CO(2) maintained at 38 mm Hg during baseline and hyperoxia. Four retinal hemodynamic measurements were acquired using bi-directional laser Doppler velocimetry and simultaneous vessel densitometry (Canon Laser Blood Flowmeter, CLBF-100, Japan) at equivalent positions on the STA and ITA. Statistical analysis was performed using linear mixed-effect models. During the hyperoxic phase, the vessel diameter (STA p=0.004; ITA p=0.003), blood velocity (STA p0.05). The magnitude of retinal arteriolar vascular reactivity in response to isocapnic hyperoxic inhaled gas challenge was not significantly different between the STA and ITA. However, the correlation analysis did not reveal a significant relationship between the percentage changes in diameter, velocity and flow of the STA and ITA and did not demonstrate equal responses from the STA and ITA to gas provocation.