The present study assessed Pavlovian eyeblink (EB) conditioning, using tones and periorbital shock as the conditioned and unconditioned stimuli (CS and US), and nictitating membrane (NM) conditioning, using tones and airpuffs as the CS and US. During each experiment, CS-evoked changes in multiple-unit activity (MUA) in the medial prefrontal cortex (mPFC) were recorded. Concomitant heart rate (HR) conditioned responses (CRs) were also recorded. A nonassociative control group received explicitly unpaired presentations of the CS and US in each experiment. Increases in both NM and EB CRs occurred over sessions in the paired, but not the unpaired, groups. Decelerative HR CRs also occurred in the eyeshock, but not the airpuff, group. Although tone-evoked increases in neuronal activity were obtained during 10 initial tone-alone presentations in all groups, this activity habituated over trials. CS-evoked increases in neuronal activity also occurred, but this activity was considerably greater in the group that received periorbital shock as the US. During subsequent extinction trials, decreases in tone-evoked neuronal activity occurred in this group, compared with the previous CS/US paired trials. CS-evoked MUA increases were minimal during all except the pretraining phase of the study in the CS/US unpaired control groups and in the paired airpuff group. These findings show that neuronal activity during associative learning occurs in the mPFC during Pavlovian EB, as well as HR conditioning, but this activity apparently reflects an affective component to learning that is only indirectly related to skeletal conditioning.