Summary Growth limitation was shown to affect the toxicity of whole organisms of Escherichia coli (strain MRE160) as determined by an LD50 method with the Porton strain of mouse. Glycerol limitation produced the most toxic organisms and limitation of sulphur the least toxic. The findings are discussed as a possible approach to the preparation of less toxic bacterial vaccines. We thank Professor P. Armitage of the London School of Hygiene and Tropical Medicine for undertaking the statistical analyses.