A culture medium for phenylalanine ammonia lyase (PAL) production in E. coli was developed following preliminary studies by means of response surface methodology (RSM). The medium components having significant effect on the production were first identified using a fractional factorial design. Then, central composite design (CCD) was utilized to optimize the medium constituents and explain the combined effects of four medium constiuents, viz., glucose, yeast extract, (NH4)2HPO4 and MgSO4. A quadratic model was found to fit the PAL production. CCD revealed that the optimum values of the test variables for PAL production were glucose 28.2 g/L, yeast extract 5.01 g/L, (NH4)2HPO4 7.02 g/L and MgSO4 1.5 g/L. PAL production of 62.85 U/g, which was in agreement with the prediction, was observed in verification experiment. In comparision to the production of basal medium, 1.8-fold increase was obtained.