In this work two Zr(IV)-porphyrins were tested as potential acetate-selective ionophores. It is shown that these compounds show increased selectivity towards acetate ion, with selectivity sequence: Cl- < NO3- < SCN- < ClO4- < acetate- < F-, when incorporated into polymeric ion-selective membranes. Among tested ionophores, Zr(IV)-tetra(tert-butylphenyl)porphyrin was found to be the best in terms of response time (20 s) and lower detection limit (2 x 10(-4) M acetate-). Designed electrodes were used as detectors in flow-injection analysis system for acetylcholine determination. Enzymatic hydrolysis was used to generate acetate ions from acetylcholine, employing acetylcholinesterase immobilized on Amberlite. The optimization of enzymatic reactions and flow-injection analysis system configuration were performed and enzyme immobilization procedures have been evaluated. It is shown that resulting flow-injection system exhibits good working parameters, such as reproducibility (standard deviation < or = 3.5%), sampling rate (60 samples per hour) and lifetime (over a week).