Extracellular levels of acetylcholine in inferior temporal cortex (IT), perirhinal cortex (PR), and dentate gyrus (DG) of the hippocampus were monitored using in vivo microdialysis in rhesus monkeys performing two behavioral tasks. Performance on a visual recognition task was associated with 26%, 41% and 24% increases in acetylcholine overflow in IT, PR and DG, respectively, compared with pre-test baseline levels. Performance on a memory-independent task was associated with increases in acetylcholine release of 24%, 34% and 7% above baseline in IT, PR and DG, respectively. The PR-DG differences were significant, but the others were not. The results provide biochemical evidence for cerebral cholinergic system activation during visually mediated behavior in non-human primates, and are consistent with the view that such activation is a prerequisite for visual recognition memory.