The role protein phosphatase 2B (calcineurin, CaN) plays in learning and memory has received a significant amount of attention due to its promotion of the dephosphorylation of 3'-5'-cyclic AMP response element binding protein (CREB). Researchers have ascertained that overexpression of CaN is associated with memory retention deficits [Foster TC, Sharrow KM, Masse JR, Norris CM, Kumar A (2001) Calcineurin links Ca(2+) dysregulation with brain aging. J Neurosci 21:4066-4073; Mansuy IM, Mayford M, Jacob B, Kandel ER, Bach ME (1998) Restricted and regulated overexpression reveals calcineurin as a key component in the transition from short-term to long-term memory. Cell 92:39-49], while CaN inhibition enhances learning and memory [Gerdjikov TV, Beninger RJ (2005) Differential effects of calcineurin inhibition and protein kinase A activation on nucleus accumbens amphetamine-produced conditioned place preference in rats. Eur J Neurosci 22:697-705; Ikegami S, Inokuchi K (2000) Antisense DNA against calcineurin facilitates memory in contextual fear conditioning by lowering the threshold for hippocampal long-term potentiation induction. Neuroscience 98:637-646]. The present study hypothesized that infusion of a CaN inhibitor (FK506) bilaterally into the olfactory bulbs of postnatal day 6 Sprague Dawley rat pups would prolong the duration of a conditioned odor preference and retard cyclic AMP response element binding protein dephosphorylation. A 2 mg/kg s.c. injection of isoproterenol (ISO, beta-adrenoceptor agonist) was paired with a 10 min exposure to peppermint and subsequently an infusion of FK506. Immunohistochemistry for phosphorylated 3'-5'-cyclic AMP response element binding protein (pCREB) revealed that unilateral infusion of FK506 resulted in an amplification of phosphorylated CREB in the olfactory bulb 40 min after training compared with saline-infused bulbs. Pups infused bilaterally with FK506 maintained a learned preference for peppermint 48, 72 and 96 h after training. CaN inhibition also modified the conventional inverted U curve obtained when ISO is used to replace stroking, as the unconditioned stimulus. When pups were infused with FK506, learning occurred with sub- and supra-optimal doses of ISO indicating that CaN overcomes non-optimal effects ISO may have on learning. We demonstrate that CaN inhibition can extend the duration of conditioned olfactory memory and may provide a target for memory prolongation that is superior to even phosphodiesterase inhibition observed in previous studies.