Several techniques for protein extraction were tested for recovering penicillin acylase from a recombinant strain of Escherichia coil. These techniques include chemical [guanidine hydrochloride, Triton X-100, ethylenediaminetetraacetic acid (EDTA), ethanol/toluene], physical (sonication, freeze-and-thawing), and enzymatic (lysozyme) treatments. Best results were obtained with the combined use of guanidine and EDTA. This extraction procedure was optimized, and it was found that 95% of the enzyme was extracted after a 10 mM EDTA plus 10 mM guanidine treatment at room temperature for 10 h. The purification factor was 25 when compared to disruption by sonication. This extraction method could avoid purification steps for particular applications.