The in vitro mouse hepatic microsomal metabolism of the macrocyclic pyrrolizidine alkaloid senecionine was studied by high-performance liquid chromatography. A muBondapak-C18 reverse-phase system was developed to study the senecionine metabolites over a wide range of polarities. Methods were further developed for the isolation of each individual metabolite. Senecic acid, senecionine N-oxide, and 19-hydroxysenecionine, a new metabolite, were isolated from the microsomal enzyme system of BALB/c mice. The metabolite, dehydroretronecine, which had previously been isolated from rat hepatic microsomes, was not detected, and minor metabolites were not identified.