Many membrane proteins such as ion channels are oligomers, but the determinants of the degree of oligomerization are not fully understood. Mechanosensitive channel with large conductance (MscL), which is ubiquitous in bacteria, is a homopentamer with two transmembrane helices and a cytoplasmic helix in each subunit. The carboxyl-terminal cytoplasmic helices assemble into a pentameric bundle that resembles cartilage oligomeric matrix protein. To address the role of cytoplasmic helices in the pentamer formation of Escherichia coli MscL, we generated MscL constructs with various deletions at the carboxyl terminus and translated them in a cell-free system. Deletions of Leu-129 and the downstream sequence resulted in formation of various oligomers without preference to pentamers, suggesting that nearly the whole cytoplasmic helix is required for MscL pentamer formation.