Calibrated functional magnetic resonance imaging can remove unwanted sources of signal variability in the blood oxygenation level-dependent (BOLD) response. This is achieved by scaling, using information from a perfusion-sensitive scan during a purely vascular challenge, typically induced by a gas manipulation or a breath-hold task. In this work, we seek for a validation of the use of the resting-state fluctuation amplitude (RSFA) as a scaling factor to remove vascular contributions from the BOLD response. Given the peculiarity of depth-dependent vascularization in gray matter, BOLD and vascular space occupancy (VASO) data were acquired at submillimeter resolution and averaged across cortical laminae. RSFA from the primary motor cortex was, thus, compared to the amplitude of hypercapnia-induced signal changes (tSD hc ) and with the M factor of the Davis model on a laminar level. High linear correlations were observed for RSFA and tSD hc (R 2 = 0.92 ± 0.06) and somewhat reduced for RSFA and M (R 2 = 0.62 ± 0.19). Laminar profiles of RSFAnormalized BOLD signal changes yielded good agreement with corresponding VASO profiles. Overall, this suggests that RSFA contains strong vascular components and is also modulated by baseline quantities contained in the M factor. We Occupancy; WM, white matter; Mathematical symbols: CBF, cerebral blood flow; CBV, cerebral blood volume; CMR O2 , cerebral metabolic rate of oxygen consumption; CVR, cerebrovascular reactivity; f, normalized CBF; M, calibration constant corresponding to the maximal BOLD signal change; P ET CO 2 , end-tidal partial pressure of carbon dioxide; P ET O 2 , end-tidal partial pressure of oxygen; R 2 , coefficient of determination; R 0 2 , reversible transverse relaxation rate; RSFA, fluctuation amplitude of the resting-state timeseries; r, normalized CMRO2; S, signal amplitude; ΔS, relative BOLD signal change; ΔS hc , relative BOLD signal change induced by mild hypercapnia; T 1 , longitudinal relaxation time; TE, echo time; TI, inversion time; TR, repetition time; tSD hc , temporal standard deviation of the hypercapnia timeseries; tSD rs , temporal standard deviation of the resting-state timeseries; tSNR, temporal signal-to-noise ratio; p, error probability; v, normalized CBV; Z, Z-score; α, Grubb exponent; β, constant describing the coupling between R 0 2 and [dHb]; κ, proportionality constant; 0 , index indicating a baseline ("resting") level; full , index indicating the entire frequency band; high, index indicating the high-frequency band; low, index indicating the low-frequency band; t, index indicating the total blood compartment; v, index indicating the venous compartment; [X], concentration of compound X.Maria Guidi and Laurentius Huber contributed equally to this study.