Alkaline phosphatase (AP) assays were performed at pH 7.5 with [N-ferrocenoyl],-6-amino-2,4-dimethylphenyl phosphate (3) as substrate and a Nafion(R)-modified glassy carbon (GC + Nafion(R)) electrode as sensor. At this pH value, the substrate was dianionic and therefore was repulsed by the GC + Nafion(R) electrode, whereas the corresponding alcohol 4 generated enzymatically was entrapped within the Nafion(R) film as a ferricinium salt by applying a potential of 0.6 V vs. AgAgCl during an accumulation step that followed incubation and preceded the electrochemical determination of 4. In a typical experiment, enzymatic hydrolysis of 3 to the corresponding alcohol 4 was carried out for 15 min in Tris buffer solution at pH 10.2. The pH value was then lowered to 7.5 and the solution was transferred to an electrochemical cell. The accumulation occurred for 5 min at a rotating GC + Nafion(R) electrode, prior to the determination of 4 by square-wave voltammetry at stationary GC + Nafion(R) electrode. The AP detection limit was 0.02 U l(-1) (signal/residual current = 2). The accumulation of [N-ferrocenoyl]-4-aminophenol (2) within the Nafion(R) film was also examined. It was observed that, at low concentrations, the dimethylated alcohol 4 accumulated more readily in the Nafion(R) film than 2, which is consistent with the more hydrophobic character of the former alcohol. Therefore the use of [N-ferrocenoyl]-4-aminophenyl phosphate as substrate for very sensitive AP assays at the Nafion(R) electrode is not recommended.