Various methods are compared for the periplasmic release of a recombinant creatinase from Escherichia coli. The examined methods include cell disruption (sonication and toluene/ethanol), chemical permeabilization (Triton X-100, guanidine-HCl, guanidine-HCl/ EDTA, chloroform, and glycine), and osmotic shock. It is found that methods involving the removal of lipopolysaccharide (such as guanidine-HCl/EDTA and osmotic shock) can achieve both excellent recovery and high purity. These findings indicate that the absence of lipopolysaccharide is closely related to the permeability for periplasmic release. Since it is based on diffusion, guanidine-HCl/EDTA achieves a much slower rate of periplasmic release than osmotic shock, which is based on shrinkage-and-swelling pumping. Osmotic shock can be further improved by using lysozyme or Ca2+-pretreatment, where the latter is seemingly the most suitable method for periplasmic release.