Two water-soluble basic antibiotics A-396-I and II were isolated from a culture broth of Streptoverticillium eurocidicus A-396. Both antibiotics gave similar analytical data indicative of a molecular formula C19±iH35±2O13N3, but different optical activities : A-396-I, |>]D +ll.6° (in water), and A-396-II, [<#]D +21.8° (in water). A comparison with known antibiotics using thinlayer chromatography suggested that A-396-I is a new antibiotic and A-396-II is identical with hygromycin B. A streptomyces strain A-396 was isolated from a soil sample collected at Shimane Prefecture. Morphological, cultural and physiological characteristics of the strain were studied and the results indicated it to be Streptoverticillium eurocidicus. When this strain was grown in suitable media produced in nearly equal amounts of two water-soluble basic antibiotics designated as A-396-I and II. Both antibiotics were isolated by adsorption procedure on ion-exchange resin and activated carbon. They were separated from each other by preparative layer chromatography on silica gel, further purified through crystalline reineckates. Finally hydrochlorides and free bases of both antibiotics were obtained.