Elevation of intracellular cyclic adenosine monophosphate (cAMP) concentrations and subsequent regulation of downstream target gene expression through phosphorylation of cAMP-responsive element binding protein (CREB) is hypothesized to underlie the mechanism(s) of long-term memory (LTM) formation. The phosphodiesterase 4 (PDE4) enzyme family is believed to play a key role in LTM by regulating cAMP levels. Thus far, four PDE4 isoforms have been identified (PDE4A, B, C and D); however, the requisite involvement of each of these isoforms in mediating LTM has yet to be elucidated. In the present study, genetic knockout mice were used to investigate the involvement of the PDE4D isoform in both in vitro and in vivo models of learning and memory. Hippocampal synaptic transmission measured electrophysiologically in CA1 slice preparations was similar between wild-type and PDE4D −/− mice yet, relative to wild-type controls, knockout mice displayed enhanced early long-term potentiation (LTP) following multiple induction protocols. Interestingly, the PDE4D −/− animals exhibited significant behavioral deficits in associative learning using a conditioned fear paradigm as compared with control littermates. The impairment in fear conditioning observed in the PDE4D −/− mice could not be attributed to differences in acquisition of the task, alterations in locomotor activity or effects on shock sensitivity. Overall, the in vitro and in vivo alterations in synaptic plasticity observed in the PDE4D −/− mice may be explained by adaptive responses occurring throughout development, and suggest that the PDE4D isoform may be an important mediator of LTM formation.