Previously syntactic irregularity has been most studied with chord sequences. However, the same chord may be interpreted as having different harmonic functions, implying perceptual ambiguity. Hence, syntactic irregularity and perceptual ambiguity may be processed simultaneously. We devised 3 different 5-chord sequences in which the ending chord differed with the tonic (T), submediant (SM), and supertonic (ST). In terms of syntactic regularity, T is most regular, ST is most irregular. However, in terms of perceptual ambiguity, the most irregular ST had the salient highest voice. Therefore, the SM was the most ambiguous condition.We investigated how the human brain separates syntactic irregularity and perceptual ambiguity in terms of effective connectivity in bilateral inferior frontal gyri (IFGs) and superior temporal gyri (STGs) with magnetoencephalography in 19 subjects. Correct rate was lower for the most ambiguous chord (SM) (P = 0.020) as expected. Connectivity from the right to the left IFG was enhanced for the most irregular chord (ST) (P = 0.024, false discovery rate (FDR) corrected), whereas connectivity from the right to the left STG was enhanced for the most ambiguous chord (SM) (P < 0.001, FDR corrected). The correct rate was negatively correlated with connectivity in the STG, further reflecting perceptual ambiguity (P = 0.026). We found that syntactic irregularity and perceptual ambiguity in music are dissociated in connectivity between bilateral IFGs and STGs, respectively.We provide the first neurophysiological evidence of the processing of perceptual ambiguity, other than syntactic irregularity, implied in musical chords. We found that the notion of "perceptually ambiguity" is applicable to musical chord stimuli different in syntactic irregularity, and that perceptual ambiguity is separate from syntactic irregularity. Our data demonstrate that the brain interprets the three conditions of musical chords as both "from regular to irregular" and "from ambiguous to unambiguous" conditions simultaneously. This study is the first to unveil dissociation of connectivity by syntactic irregularity and perceptual ambiguity involved in musical chord stimuli.