Enacyloxins (RNXs) are antibiotic metabolites produced by Frateuria sp. W-315 cultured in the spent medium of N. crassa, and were shown to be active against Gram-positive and Gram-negative bacteria. The chemical structure of ENX IIa, the main component and the most active congener among ENXs, was studied via spectroscopic and degradative works. It was elucidated as a novel structure of (1S,3R,4S,2'E,4'E,6'E,8'E,10'Z,20'E)-3-(19'-carbamoyloxy-11',18'-dichloro-6',12'-dimethyl-15'-oxo-13',14',17'-trihydroxytricosa-2',4',6',8',10',20'-hexaenoyloxy)-4-hydroxy-1-cyclohexanecarboxylic acid. ENX IVa, isolated as a biosynthetic intermediate of ENX IIa, was determined be 15'-hydroxy analog of ENX IIa. Conversion of ENX IVa to ENX IIa was catalyzed by the soluble enzyme present in the culture medium of Frateuria. The enzyme exhibited the oxidative activity even in a refrigerator. At room temperature, ENX IVa in the culture supernatant encountered the enzymatic hydrolysis of carbamoyl moiety as well as the above mentioned oxidation. Factors concerning the production of ENXs in the spent medium of N. crassa were also investigated.