Acetylcholine (Ach) is believed to play a major role in attentional processing in cortex, thereby contributing to memory formation and maintenance. To understand its role in these neuronal processes, we set out to monitor ACh level-changes in prefrontal cortex of a monkey performing a delayed match to sample task. The changes were monitored during a phase of rest and during the task to compare the baseline level of Ach with the level during task. We used a push and pull method to sample extracellular brain fluid (EBF) from the prefrontal cortex (PFC) at very low flow rates, low nanol/min range. These flow rates were chosen to reduce the depletion of the neuronal environment in contrast to the conventionally used microdialysis. After the sampling of the EBF, the capillary, where the EBF samples were stored, was directly coupled to capillary high performance liquid chromatography-mass spectrometry (HPLC-MS) for the chemical analysis to detect and quantify ACh. The combination of the sampling and the analysis system proofed possible to determine amounts of Ach in the attomole range from the EBF samples. In the experiments a 7-min interval was chosen to monitor the Ach changes during rest and the task. We could reliably detect and quantify Ach concentrations during rest and the delayed match to sample task, showing a significant increase in Ach concentration during the task. Our results show clearly the involvement of acetylcholine in behaving monkeys involved in a delayed match to sample task, where memory formation and attention are mandatory for the appropriate performance during the task.