Confocal laser scanning microscopy was applied to measure the pH value in the carrier of immobilized enzymes during the enzyme-catalyzed synthesis. pH profiles with a high resolution are shown, with the pH increasing in the core of the particles. Significant differences occur for different carrier material, particle size, porosity and surface modification. The increased pH value is identified as one of the reasons leading to reduced enzyme selectivity in the penicillin amidase catalyzed synthesis of cephalosporins and penicillins.