An electrochemical flow analysis system was optimized together with immobilized putrescine oxidase and horseradish peroxidase for putrescine measurement. Four coupling agents, suberic acid bis(N-hydroxysuccinimide ester), gamma-maleimidobutyric N-hydroxysuccinimide ester, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and glutaraldehyde, were used for immobilizing the two enzymes on porous aminopropyl glass beads to form a bienzymic detection column. Although the glutaraldehyde crosslinking procedure offered the highest response, the immobilized bienzyme system was responsive to putrescine, spermidine (123% of the putrescine response at 250 muM) and cadaverine (98% of the putrescine response). In contrast, the enzymes immobilized on the glass beads using suberic acid bis(N-hydroxysuccinimide ester) offered significantly better selectivity towards putrescine at the same concentration. For comparison, cadaverine and spermidine only provoked a response of 4.7% and 27.5% of the putrescine signal. The response to cadaverine and spermidine was further suppressed by lowering the detection pH from 8 to 7. At 250 muM, the response obtained for cadaverine and spermidine was only 1.5% and 3.9%, respectively, of the signal obtained for putrescine. A linear response to putrescine was obtained from 5 to 75 muM (0.629 muAs muM-1, R2 = 0.997) with a detection limit of 5 muM (S/N = 3). The amperometric response retained 75% of its initial value after 600 repeated injections. The immobilized PUO/HRP (putrescine oxidase/horseradish peroxidase) was successfully demonstrated for measuring putrescine in fish extracts as an indicator of fish spoilage.