The cyclic AMP-dependent protein kinase (PKA) signaling pathway has been shown be important in mechanisms of synaptic plasticity but its direct and downstream signaling effects are not well understood. Using an in vitro model of eyeblink classical conditioning, we report that PKA has a critical role in initiating a signaling cascade that results in synaptic delivery of GluR1- and GluR4-containing AMPARs during conditioning. PKA and the Ca²⁺-calmodulin-dependent protein kinases (CaMKs) II and IV are activated early in conditioning and are required for acquisition and expression of conditioned responses (CRs). CREB is also activated early in conditioning but is blocked by coapplication of inhibitors to PKA and the CaMKs suggesting that CREB is downstream of those signaling cascades. Moreover, evidence suggests that PKA activates extracellular signal-regulated kinase (ERK) which is also required for conditioning. Imaging studies after conditioning further indicate that colocalization of GluR1 AMPAR subunits with the synaptic marker synaptophysin requires PKA but is insensitive to the NMDAR inhibitor AP-5. PKA activation also leads to synaptic localization of GluR4 subunits that, unlike GluR1, is dependent on NMDARs and is mediated by CaMKII. Together with previous studies, our findings support a two-stage model of AMPAR synaptic delivery during acquisition of classical conditioning. The first stage involves synaptic incorporation of GluR1-containing AMPARs that serves to activate silent synapses. This allows a second stage of NMDAR- and PKC-dependent delivery of GluR4 AMPAR subunits that supports the acquisition of CRs.