Effluents from cat paws perfused with compound 48/80 contained histamine and lipid-soluble smooth muscle stimulating principles. Fractionation of the lipid-soluble material on silicic acid resulted in a separation into two major components with different biological properties. One component was of polar character and elicited a slow, sustained contraction of the isolated guinea-pig ileum. These are effects associated with “slow reacting substance” (SRS) by definition. The other component being less polar was examined by a variety of chromatographic procedures in combination with bioassay. In silicic acid chromatography with linear gradient elution and in reversed phase partition chromatography all biological activity followed the radioactivity due to added 3H-labelled prostaglandin E2 (PGE2). On thin layer chromatography the smooth muscle stimulating compound cochromatographed with reference PGE2. When the spasmogenic compound isolated by silicic acid chromatography was incubated with 15-hydroxy prostaglandin dehydrogenase from swine lung in the presence of NAD+ complete biological inactivation occurred. Determination of ester-bound C20 fatty acids in cat skin by gas chromatography revealed that arachidonate, precursor of PGE2, was the most abundant prostaglandin precursor acid. When 3H-PGE2 was infused into cat paws the parent compound as well as two metabolites, 13,14-dihydro-PGE2 and 15-keto-13,14-dihydro-PGE2, appeared in the effluent. It is concluded that PGE2 constitutes part of the lipid-soluble spasmogenic material appearing in connection with histamine release in the cat paw induced by compound 48/80.