Selective amperometric enzyme microsensors for monitoring low micromolar concentrations of choline in extracellular fluid of rat brain have been developed. Preparation of the choline microsensors involved the modification of carbon fiber microcylinder electrodes (10 microns diameter, 300-400 microns long) with a cross-linked redox-active gel containing horseradish peroxidase and choline oxidase. Rejection of the noise recorded from the choline microsensors implanted in living brain tissue improved the in vivo detection capabilities of the sensors. The microsensors and a differential detection scheme were used to estimate the basal concentration of choline in striatal tissue at 6.6 +/- 2.9 microM and to measure changes in choline concentrations of 6.1 +/- 2.7 microM in vivo. The microsensors were also used to monitor choline produced following the injections of acetylcholine in vivo. Coinjections of neostigmine and acetylcholine significantly lowered the choline response recorded with the microsensors, confirming that the response following the injections of acetylcholine alone was due to the activity of endogenous acetylcholinesterase. Comparison of the maximal rate of decrease in choline concentration following the injections of 1 mM choline and 1 mM acetylcholine was used to estimate the rate of acetylcholine clearance from extracellular fluid through cholinesterase activity at approx. 2.5 microM/min.