In in vivo studies, a conditioned increase in NK cell activity can be obtained by pairing odor of camphor (conditioned stimulus, CS) with poly I:C (unconditioned stimulus, US) in a single-association paradigm. We identified interferon (IFN) as the signal that reaches the central nervous system (CNS) to make an association with the camphor CS. We have also established that the CS/US association is an IFN-dependent step, and the expression stage is an opioid-dependent pathway which can be blocked with naltrexone and dexamethasone. Here we have focused on the signals responsible for the expression of conditioned augmentation of natural killer (NK) cell activity. The possible efferent signal molecules that were considered were IFN, beta-endorphin (beta-END), and adrenocorticotropic hormone (ACTH). Plasma levels of beta-END and ACTH of conditioned and control mice were quantitated by radioimmunoassay, and the changes in IFN message in the spleen cells were determined by Northern hybridization analysis. Results indicate that the ACTH levels and IFN-alpha gene expression were higher in the conditioned animals than in the controls. These studies support the view that ACTH released from the pituitary gland is involved in the up-regulation of IFN-alpha, which in turn stimulates the NK cells in the spleen.