A new peptide antibiotic, brevsitin, was isolated from the culture broth of B. brevis 342-14. Brevistin is an acylpeptide with an approximate empirical formula of C63H91N15O18 containing aspartic acid, threonine, glycine, valine, isoleucine, phenylalanine, tryptophan and 2,4-diaminobutyric acid. The antibiotic is soluble in acid and alkaline water. Its hydrochloride is soluble in methanol. Brevistin is active against gram-positive bacteria in vitro and in vivo, and is of low toxicity to mice.