Carbon dioxide is a potent cerebral vasodilator. We have identified a significant source of low-frequency variation in blood oxygen level-dependent (BOLD) magnetic resonance imaging (MRI) signal at 3 T arising from spontaneous fluctuations in arterial carbon dioxide level in volunteers at rest. Fluctuations in the partial pressure of end-tidal carbon dioxide (P et CO 2 ) of ±1.1 mm Hg in the frequency range 0–0.05 Hz were observed in a cohort of nine volunteers. Correlating with these fluctuations were significant generalized grey and white matter BOLD signal fluctuations. We observed a mean (±standard error) regression coefficient across the group of 0.110 ± 0.033% BOLD signal change per mm Hg CO 2 for grey matter and 0.049 ± 0.022% per mm Hg in white matter. P et CO 2 -related BOLD signal fluctuations showed regional differences across the grey matter, suggesting variability of the responsiveness to carbon dioxide at rest. Functional magnetic resonance imaging (fMRI) results were corroborated by transcranial Doppler (TCD) ultrasound measurements of the middle cerebral artery (MCA) blood velocity in a cohort of four volunteers. Significant P et CO 2 -correlated fluctuations in MCA blood velocity were observed with a lag of 6.3 ± 1.2 s (mean ± standard error) with respect to P et CO 2 changes. This haemodynamic lag was adopted in the analysis of the BOLD signal. Doppler ultrasound suggests that a component of low-frequency BOLD signal fluctuations is mediated by CO 2-induced changes in cerebral blood flow (CBF). These fluctuations are a source of physiological noise and a potentially important confounding factor in fMRI paradigms that modify breathing. However, they can also be used for mapping regional vascular responsiveness to CO 2.