A highly sensitive enzyme immunoassay (EIA) system for quantitative determination of benzoylmesaconine (BM) in biological samples was developed. The sensitivity of an antiserum towards β-galactosidase (β-gal)-labeled antigen was in the range of 10−1–105 ng/ml of BM in rat serum. Under the same conditions, the antiserum had quite weak cross-reactivity with related alkaloids in the aconite tuber. The coefficients of variance (CV) of intra- and interday assays were 6.4 and 10.2%, and 3.4 and 14.8% in the presence of high and low concentrations, respectively, of BM in rat serum. The concentration versus time curve of BM after intravenous administration corresponded to a two-compartment model, and that after oral administration corresponded to a one-compartment model. Benzoylmesaconine was identified as two peaks in serum at 48 h after oral administration; consequently its area under the serum concentration–time curve (AUC0–48) and mean residence time (MRT) values were significantly high. Hydrolysis by intestinal bacteria followed by intestinal reabsorption accounted for these values. One hour after the oral administration of 0.8, 0.2 and 0.05 mg/kg of aconitine (A) or mesaconitine (M), the concentrations of each and their respective metabolites benzoylaconine (BA) or BM in serum and spinal cord samples were investigated. The concentrations of the original compounds were lower than those of their metabolites at all three doses, but the increase in metabolites was more obvious in the spinal cord than in serum.