<jats:p><jats:bold>Abstract:</jats:bold> FR134043 is a semisynthetic disulfonated derivative of the natural product FR901277, is isolated from the culture filtrate of <jats:italic>Streptomyces resistomicificus</jats:italic> and has potent inhibitory activity against human leukocyte elastase. Although the chemical structure of FR134043 was determined to be a unique bicyclic peptide‐like compound consisting of seven amino acids by using several spectroscopic analytical methods, the chiralities of three centers were unknown. A simple simulated annealing protocol to determine the structure was applied to the eight possible stereoisomers, and the one that best satisfied the NOE distance constraints was determined to be the true stereoconfiguration of FR134043. The solution structure showed that all Cα atoms existed in the <jats:sc>l</jats:sc> configuration and six of the seven side chains were located towards the outside of the bicyclic framework, even though most of them are highly hydrophobic moieties. The simulated annealing calculation described here is a frequently used method for the determination of the solution structure of peptides or small proteins. We show here that it is also applicable to the determination of the absolute configuration of macrocyclic compounds produced from natural sources.