We used a high-resolution confocal scanning laser ophthalmoscope to measure the magnitude of change in retinal arteriolar diameters in response to oxygen breathing in young, healthy nonsmokers and smokers. Image sequences were obtained before and during oxygen breathing. Image sequences were desinusoided, registered, and averaged, before vessel diameters were measured using a sliding linear regression filter. Arteriole diameters were observed to constrict during the first 5 min. of oxygen breathing, plateau, and remain stable while hyperoxia was maintained, returning to baseline at the end of the hyperoxic period. Blood flow to the temporal retina was found to be higher than to the nasal retina (p=0.008). The percentage constriction of vessels did not vary across retinal quadrants (p=0.372, analysis of variance) and did not depend on vessel size (p=0.538). Baseline diameters were unaffected by acute cigarette smoking. The magnitude of vasoconstriction was diminished in smokers compared to nonsmokers (p=0.017), while acute smoking did not influence the percentage constriction attained by the vessels (p=0.621). Using a high-resolution imaging technique allowed us to measure reactivity to a high degree of accuracy and to assess it in vessels of smaller caliber than were previously studied.