Head-shake response (HSR) habituation was presently used to investigate the phenomena of spontaneous recovery and neural plasticity. Independent groups of rats were presented with five consecutive habituation sessions separated by inter-session intervals (ISIs) of 2, 24 or 72 h. At the conclusion of testing hippocampus and prefrontal cortex tissue samples were collected for determination of matrix metalloproteinase-3 (MMP-3:stromelysin-1) expression as a marker of neural plasticity. The results indicated that by the fifth session the 2 h ISI group showed no spontaneous recovery, the 72 h ISI group revealed nearly complete spontaneous recovery; while the 24 h ISI group showed intermediate recovery. MMP-3 expression in the hippocampus and prefrontal cortex was elevated in the 2 and 72 h ISI groups, but not in the 24 h group. A second experiment utilized 7-day osmotic pumps to intracerebroventricularly infuse an MMP-3 inhibitor for 6 days. The animals were then tested on the seventh day using the 2 h ISI protocol. Delivery of the MMP-3 inhibitor facilitated spontaneous recovery, thus compromising the animal's ability to appropriately habituate. This effect was accompanied by a significant inhibition of hippocampus and prefrontal cortex MMP-3 expression. These results suggest that elevations in hippocampus and prefrontal cortex MMP-3 expression contribute to this simplest form of learning and may be a mechanism underlying spontaneous recovery.