Bacterial L-forms are cell wall-less forms of bacteria that usually grow with a conventional cell wall. Despite being important for research, L-forms are difficult to generate reproducibly and research in this area is challenging. Domínguez-Cuevas et al. (2011) report a method to rapidly, quantitatively and reproducibly generate populations of L-forms in Bacillus subtilis. Importantly, the methodology may be applicable to other bacteria heralding a new era of L-form research. Moreover, the genetic requirements of this method provide insights into how Lipid II synthesis and autolysin expression/activity are normally balanced and the central role of the WalRK two-component system in this process. © 2011 Blackwell Publishing Ltd.