A highly sensitive and specific competitive enzyme-linked immunoassay (ELISA) for the determination of retrorsine, senecionine, and integerrimine in the nanogram range has been developed. Pyrrolizidine alkaloids were detected in Senecio rupestris. No cross-reactivity was seen with either retrosine N-oxide, otonecine alkaloids, or other macrocyclic pyrrolizidine alkaloids.