In our screening studies of peptide antibiotics produced by soil bacteria, a new antibiotic complex, polymyxin P, was isolated from a culture of strain T-39 (identified as Bacillus polymyxa) obtained from soil collected at Suma-ku, Kobe City, Japan. General properties of the antibiotic complex resemble those of known polymyxins, but obvious differences were found by chromatography and amino acid content of the hydrolyzate. Thus, it was concluded to be a new member of the polymyxin group and named polymyxin P. The acetate of polymyxin P showed antimicrobial activity against Gram-negative bacteria such as Escherichia coli NIH T, Salmonella typhosa (MIC 0.78 mcg/ml), etc., but was ineffective against Gram-positive bacteria and Candida albicans. Further purification by countercurrent distribution separated it into two active components, Px and P2, whose amino acid constituents were the same as polymyxin P but contained 6-methyloctanoic acid and isooctanoic acid, respectively. The fatty acids of Px and P2 were identical with those of corresponding components of known polymyxins.