Synthesis of cephalexin with immobilized penicillin acylase at high substrates concentration at an acyl donor to nucleophile molar ratio of 3 was comparatively evaluated in aqueous and ethylene glycol media using a statistical model. Variables under study were temperature, pH and enzyme to substrate ratio and their effects were evaluated on cephalexin yield, ratio of initial rates of cephalexin synthesis to phenylglycine methyl ester hydrolysis, volumetric and specific productivity of cephalexin synthesis, that were used as response parameters. Results obtained in both reaction media were modeled using surface of response methodology and optimal operation conditions were determined in terms of an objective function based on the above parameters. At very high substrates concentrations the use of organic co-solvents was not required to attain high yields and actually almost stoichiometric yields were obtained in a fully aqueous media with the advantages of higher productivities than in an organic co-solvent media and compliance with the principles of green chemistry.