Numerous recombinant proteins of industrial and pharmaceutical importance are secreted to the periplasmic space of Escherichia coli; yet, very few generic periplasmic recovery methods with the potential for scaleup exist This work describes the development of an enzymatic method for the release of recombinant proteins from the periplasm of E. coli. The simple two-step method involving resuspension of the cells in a fractionation buffer, followed by recovery of the periplasmic fraction, has been shown to be feasible for use in both the laboratory and 5-1 scale. For the efficient release of a recombinant Streptomyces thermoviolaceus alpha-amylase from the E. coli periplasm, it has been shown that an osmotic shock must immediately follow lysozyme treatment to obtain high yields. The phase of growth and level of recombinant protein expression has an affect on the recovery from the E. coli periplasm. Yields of 70-90% of recombinant alpha-amylase were recovered from the periplasm of stationary phase E. coli cells in both laboratory and 5-1 scale experiments.