An innovative approach to determine the orientation of penicillin G acylase (PGA) from Escherichia coli covalently immobilized onto solid supports has been developed. This method is based on tryptic digestion of immobilized PGA followed by HPLC-MS analysis of the released peptides which are supposed to be only those exposed toward the reaction medium and not directly bound to the solid support. To this purpose, PGA was immobilized on Eupergit C (acrylic hydrophobic resin) and glyoxyl-agarose (hydrophilic resin) functionalized with epoxy and aldehyde groups, respectively, both involving the Lys residues of the protein. The peptide maps obtained were analyzed to derive the orientation of immobilized PGA, as the position of the detected Lys gave indication concerning the accessibility of the different areas of the protein. The results indicate that PGA immobilization on both supports involves mainly Lys located near the binding pocket (70%). Some differences in the enzyme orientation on the two supports can be deduced by the presence of different unbound Lys residues in the released peptides, specific to each support (Lys 117 alpha for PGA-Eupergit C; Lys 163 alpha and Lys 165 alpha for PGA-glyoxyl-agarose). These results have been correlated with the data obtained in the kinetically controlled synthesis and indicate that the orientation of PGA on both supports is partially unfavorable, driving the active site near the support surface. This type of orientation of the enzyme enhances the effect of the nature of the support and of the binding chemistry on the catalytic properties. The information obtained indicated the most suitable support and activation strategy to design an immobilized acylase with good synthetic properties for preparative processes. The glyoxyl-Eupergit C support with enhanced porosity synergically combines the mechanical stability and synthetic performances of immobilized PGA and was successfully used in the synthesis of several cephalosporins.