Neuronal activity causes changes in both cerebral metabolic rate of oxygen (CMRO2) and cerebral blood flow (CBF). Since the relationship between tissue oxygenation and regional CBF (rCBF) during exercise has not been elucidated, we compared the data obtained using near-infrared spectroscopy (NIRS) and rCBF examined using positron emission tomography (PET). Participants in this study comprised 26 healthy young men. Changes in concentration of oxygenated hemoglobin (ΔO2Hb) and deoxygenated hemoglobin (ΔHHb) in the prefrontal cortex (PFC) were measured using NIRS continuously during a 15-min bout of the constant-load low-intensity cycling exercise (n = 14). Under the same protocol as the NIRS study, rCBF was measured using H215O and PET by the autoradiographic method at baseline (Rest) and at 3 min (Ex1) and 13 min (Ex2) after starting exercise (n = 12). As systematic factors influenced by exercise, heart rate, end-tidal pressure of carbon dioxide (PETCO2) and blood pressure (BP) were monitored. For each region investigated by NIRS, rCBF was analyzed quantitatively using PET-MRI co-registered standardized images. Despite inter-individual differences, changing patterns of ΔO2Hb and ΔHHb in the PFC were similar between channels. Significant main effects for time point were identified in ΔO2Hb, ΔHHb and changes in rCBF. While rCBF increased from rest, ΔO2Hb was not changed at Ex1. Conversely, rCBF was unchanged from rest but ΔO2Hb was significantly increased at Ex2. Fluctuations of PETCO2 and BP evoked by exercise were not in accordance with changes in ΔO2Hb, ΔHHb and rCBF, while BP may affect the forehead skin blood flow. Given that NIRS data are a mixture of skin and brain effects, our results suggest that CMRO2 may differ between the phases in a bout of dynamic exercise. The present study indicates the utility of NIRS to examine the relationship between CMRO2 and rCBF during exercise.