Summary Many bacteria are able to assume a transient cell wall‐deficient (or L ‐form) state under favourable osmotic conditions. Cell wall stress such as exposure to β‐lactam antibiotics can enforce the transition to and maintenance of this state. L ‐forms actively proliferate and can return to the walled state upon removal of the inducing agent. We have adopted E scherichia coli as a model system for the controlled transition to and reversion from the L ‐form state, and have studied these dynamics with genetics, cell biology and ‘omics’ technologies. As such, a transposon mutagenesis screen underscored the requirement for the R cs phosphorelay and colanic acid synthesis, while proteomics show only little differences between rods and L ‐forms. In contrast, metabolome comparison reveals the high abundance of lysophospholipids and phospholipids with unsaturated or cyclopropanized fatty acids in E . coli L ‐forms. This increase of membrane lipids associated with increased membrane fluidity may facilitate proliferation through bud formation. Visualization of the residual peptidoglycan with a fluorescently labelled peptidoglycan binding protein indicates de novo cell wall synthesis and a role for septal peptidoglycan synthesis during bud constriction. The DD ‐carboxypeptidases PBP5 and PBP6 are threefold and fourfold upregulated in L ‐forms, indicating a specific role for regulation of crosslinking during L ‐form proliferation.