Lasso peptides exist naturally in a threaded state as [1]rotaxanes, and we reasoned that lasso peptides cleaved in their loop region could serve as building:blocks for catenaries. Mutagenesis of the lasso peptide microcin J25 (MccJ25) with two cysteine residues followed by cleavage of the peptide with trypsin. led to a [2]rotaxane structure that self-assembled into a [3]catenane and [4]catenanes at room temperature in aqueous solution. The [3]catenane represents the smallest ring size of a catenane composed solely of polypeptide segments. The NMR structure of the [3]catenane was determined, suggesting that burial of hydrophobic residues may be a driving force for assembly of the catenane structure.