The frequency of fibrillatory waves (f-waves), Ff, exhibits significant variation over time, and previous studies suggest that some of this variation is related to respiratory modulation through the autonomic nervous system. In this study, we tested the hypothesis that this variation (?Ff) could be related to acetylcholine concentration ([ACh]) release pattern. Electrocardiograms were recorded from seven patients during controlled respiration before and after full vagal blockade, from which f-wave frequency modulation was characterized. Computational simulations in human atrial tissues were performed to assess the effects of [ACh] release pattern on Ff and compared to experimental results in humans. A cross-stimulation protocol was applied onto the tissue to initiate a rotor while cyclically varying [ACh] following a sinusoidal waveform of frequency equal to 0.125 Hz. Different mean levels (0.05, 0.075µM/l) and peak-to-peak ranges (0.1, 0.05, 0.025 µM/l) of [ACh] variation were tested. In all patients, an f-wave frequency modulation could be observed. In 57% of the patients, this modulation was significantly reduced after vagal blockade. Simulations confirmed that rotor frequency variations followed the induced [ACh] patterns. Mean Ff was dependent on mean [ACh] level, while?Ffwas dependent on [ACh] variation range.