Cat paws were perfused with a physiological salt solution containing compound 48/80. In addition to histamine, a principle producing a slow sustained contraction of the isolated guinea-pig ileum appeared in the effluent. This slow reacting substance (SRS) was purified by ethanol extraction, solvent partition, silicic acid and anion exchange chromatography. The overall recovery was 10–20 per cent, calculated from the non-histamine smooth muscle stimulating activity of the ethanolic extract. The purification calculated from the crude lipid extract was about 200-fold. The purified material was dialysable, thermostable at neutral pH and more labile in acid than in alkaline milieu. It behaved homogenously on thin layer chromatography in several systems. The most marked biological effects were contraction of the isolated guinea-pig ileum and the human bronchus, increases in the bronchial resistance in guinea-pigs in vivo, in the colouration (Evan's blue dye i.v.) of guinea-pig skin after intradermal injection and in the blood flow in the cat hindlimb after intraarterial injection. Treatment of the purified material with N, N'-carbo-di-p-tolylimide, phenyl isocyanate, iodine monobromide, potassium permanganate or acetic anhydride abolished or greatly reduced the biological activity. Incubation of SRS with 15-hydroxy prostaglandin dehydrogenase in the presence of NAD+ resulted in no loss of biological activity. Together the results indicate that SRS is a biologically active carboxylic acid with hydroxyl groups and one or more double bonds but probably of non-prostaglandin nature.