Enzymatic production of cephalexin using immobilized penicillin G acylase was studied. The factors that affect the conversion of 7-amino-3-deacetoxycephalosporanic acid (7-ADCA) to cephalexin, such as pH; temperature; concentrations of 7-ADCA, D(-)-phenylglycine methyl ester (PGME), and immobilized penicillin G acylase (IMPGA); time; and molar ratios of 7-ADCA and PGME, were investigated. Under optimized conditions, 85% conversion of 7-ADCA to cephalexin was achieved. The IMPGA was used for 10 cycles.