Purpose. To compare the impact of three different techniques used to induce hyperoxia on end-tidal CO2 (PETCO2). The relationship between change in PETCO2 and retinal hemodynamics was also assessed to determine the clinical research relevance of this parameter. methods. The sample comprised 10 normal subjects (mean age, 25 years; range, 21–49 years). Each subject attended for three sessions. At each session, subjects initially breathed air followed by O2 only; O2 plus CO2, using a nonrebreathing circuit (with CO2 flow continually adjusted to negate drift of PETCO2); or air followed by O2, using a sequential rebreathing circuit. In addition, using a separate sample of eight normal subjects (mean age, 26.5 years; range, 24–36 years), a methodology that initially raised PETCO2 and then returned to homeostatic levels was used to determine the impact, if any, of perturbation of PETCO2 on retinal hemodynamics. results. The difference in group mean PETCO2 between baseline and elevated O2 breathing was significantly different (t-test, P = 0.0038) for O2-only administration with a nonrebreathing system. The sequential rebreathing technique resulted in a significantly lower difference (i.e., before and during hyperoxia) of individual PETCO2 (t-test, P = 0.0317). The PETCO2 perturbation resulted in a significant (P < 0.005) change of retinal arteriolar diameter, blood velocity, and blood flow. conclusions. The sequential rebreathing technique resulted in a reduced variability of PETCO2. A relatively modest change in PETCO2 resulted in a significant change in retinal hemodynamics. Rigorous control of PETCO2 is necesssary to attain standardized, reproducible hyperoxic stimuli for the assessment of retinal vascular reactivity.