The chemical constituents of Aconitum yesoense var. macroyesoense and Aconitum japonicum were examined using high-resolution spectral analysis. Twelve novel alkaloids were isolated from A. yesoense var. macroyesoense together with 20 known alkaloids. Eight novel alkaloids were isolated from A. japonicum together with 15 known alkaloids. An HPLC-atmospheric pressure chemical ionization-mass spectrometry (HPLC-APCI-MS) method was useful for the simultaneous determination of 21 Aconitum alkaloids found in A. yesoense var. macroyesoense and A. japonicum. These compounds were fairly stable under the conditions used, and the protonated molecules or fragment ions characteristic of the molecule appeared as base peaks in the mass spectra and were used for selected ion monitoring. HPLC-APCI-MS is a very promising approach for structural investigations of positional isomers and stereoisomers. This method was applied successfully to stereoisomeric Aconitum alkaloids differing in configuration at C-1, -6, or -12. Comparison of the APCI spectra showed that the abundance of fragment ions was significantly higher for the C-1, -6, or -12 β-form alkaloid than for C-1, -6, or -12 α-form alkaloid. The main alkaloid constituents in the root of A. yesoense var. macroyesoense, Aconitum alkaloids of the C₂₀-diterpenoid type, kobusine and pseudokobusine, and their acyl derivatives were examined for their peripheral vasoactivities by measuring laser-flowmetrically the cutaneous blood flow in the hind foot of mice after intravenous administration. It is thought that the hydroxyl groups of alkaloids, especially a free OH group of pseudokobusine at C-6, were important for action on the peripheral vasculature leading to dilatation, and the results indicated that esterification of the hydroxyl group at C-15 with either anisoate, veratroate, or p-nitrobenzoate may contribute to enhancement of the activity of the parent alkaloids.