Abstract: Two strategies were developed to synthesize the acylated cyclic peptides know as polymyxins. Synthesis of polymyxin E1 and several analogs enabled us to evaluate the minimum inhibitory concentration of individual compounds against Gram-negative bacteria. In this study we also report the first identification of two component peptides in the complex polymyxin fermentation product colistin, a Thr2Ser isoform and an acyl group isomer. Both of these peptides, as well as a known component peptide, Leu7Ile, were similar to polymyxin E1 in potency, suggesting that conservative mutations in the colistin family are functionally inconsequential. In contrast, the acyclic analogs of all of these peptides were inactive, indicating that the characteristic lariat structure of the polymyxins is necessary for antimicrobial activity.