The immobilization of an enzyme on solid support can give a catalyst with improved stability and catalytic properties compared to the native enzyme; in this work the enantioselectivity of penicillin G acylase (PGA - EC 3.5.1.11) isolated from different microbial sources (Escherichia coli, Kluyvera citrophila and Arthrobacter viscosus) has been studied. Two matrixes (Eupergit C and agarose gel beads) and different binding techniques have been investigated measuring the enantioselectivity of the different immobilized enzyme in the hydrolytic resolution of racemic esters and amides of mandelic acid. We have found a relevant dependence of the catalytic properties on the enzymatic source, immobilization technique and substrate structure. The best result has been obtained using the acylase from E. coli immobilized on glyoxyl agarose by multipoint interaction in the hydrolysis of mandelic acid iso-propylamide at pH 6.5 and 4degreesC (73% enantiomeric excess at 47% of conversion). These results suggest that the immobilization of the PGA from E. coli on different supports (via different orientations, with different rigidity) may be a useful tool to modulate the catalytic properties.