An enzyme electrode based on a glass electrode for measuring penicillin-V concentration is described. In preparing this electrode, penicillinase is immobilized by cross-linking with a very fine film of glutaraldehyde, which is sprayed directly over the sensitive end of a pH glass electrode. The penicillin sensor was used for the determination of penicillin-V concentrations in phosphate buffer, fermentation broth and dairy milk, the ranges of concentrations detected being 1.5-15, 2.5-15 and 2-12 mM, respectively. The linearity, stability, sensitivity, reversibility and pH and buffer strength dependence of the electrode were examined and are discussed. An extremely short response time of less than 10 s is obtained with electrodes made in this manner.