The activity of the septo-hippocampal and nucleus basalis-cortical cholinergic pathways was investigated by measuring changes in the extracellular acetylcholine levels in the hippocampus and parietal cortex, by means of transversal microdialysis, during the acquisition and recall of a positively reinforced operant behavior. Adult male Wistar rats were trained in a sound-isolated operant chamber equipped with a single lever. The positive reinforcement was represented by food pellets and the number of cumulative reinforced responses was recorded every 30 min. Five groups of rats were used. Unoperated animals were used as controls. In two groups of untrained animals, the microdialysis tubes were transversally implanted in the parietal cortex, and hippocampus and the training in the operant behavior chamber began 24 h after surgery. In two further groups the microdialysis tubes were implanted in the parietal cortex, and hippocampus after training for 15 days in the operant chamber. Food was removed 12 h before training. The time needed by the control rats to reach a stable baseline of reinforced responses was 83 +/- 12 min, while in the untrained rats implanted with dialysis probes in the cerebral cortex and in the hippocampus was 621 +/- 129 and 521 +/- 126 min, respectively, and in those pretrained and implanted in cerebral cortex and in the hippocampus was 116 +/- 38 and 217 +/- 59 min, respectively. In the untrained operated rats, both cortical and hippocampal extracellular acetylcholine levels remained constant until the number of reinforced responses was low but increased significantly (+156% in the cortex and +183% in the hippocampus) in the first 30 min period in which there was a sharp rise in the reinforced responses. In the pretrained operated rats, neither in the cortex nor in the hippocampus was the increase in response rate accompanied by a statistically significant increase in extracellular acetylcholine levels. Our findings demonstrate that activation of the forebrain cholinergic pathways occurs during the acquisition of a rewarded operant responses, while recall of the same behavior is not associated with the activation of the cholinergic system.