Rats were injected with the pyrrolizidine alkaloids heliotrine, indicine, or anacrotine, and killed after 20 hr. Alkaloid metabolites conjugated to haemoglobin thiol groups were recovered in the form of pyrrolic monoethyl ethers, by treating blood samples with ethanolic silver nitrate under "buffered" conditions. Chemically prepared putative toxic metabolites of the alkaloids--dehydroheliotrine, dehydroindicine, and dehydroanacrotine--were also allowed to react in vitro with blood and with an immobilized thiol, thiol-Sepharose, and subsequently the S-conjugated pyrroles were again recovered as ethyl ethers. The recovered pyrrolic ethers were identified by comparing them with reference compounds prepared from ethanol and the dehydro-alkaloids, and the structures of the S-bound pyrroles were deduced. Blood from rats given the 9-monoester alkaloids heliotrine or indicine contained pyrrolic residues, S-bound at their 9-position. Anacrotine-treated rats yielded two diastereomeric 7-ethers, showing that dehydrocrotanecine 7-conjugates had been present in the blood. The products from alkaloid-treated rats were identical with those from blood or thiol-Sepharose treated with the corresponding dehydro-alkaloids in vitro. This supported the view that proximal metabolites leading to S-binding in vivo were the dehydro-derivatives of the alkaloids. In each case the thiols were attacked by the most reactive centre of the dehydro-alkaloid: the 9-ester in dehydroheliotrine and dehydroindicine, and the 7-ester in dehydroanacrotine. Accordingly, simple chemical reactions could account for the products formed in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)