Acquisition of trace eyeblink conditioning involves the association of a conditioned stimulus (CS) with an unconditioned stimulus (US) separated by a stimulus-free trace interval. This form of conditioning is dependent upon the hippocampus and the caudal anterior cingulate cortex (AC), in addition to brain stem and cerebellar circuitry. Hippocampal involvement in trace eyeblink conditioning has been studied extensively, but the involvement of caudal AC is less well understood. In the present study, we compared neuronal responses from rabbits given either paired (trace conditioning) or unpaired (pseudoconditioning) presentations of the CS and US. Presentation of the CS elicited significant increases in neuronal activity at the onset of both trace conditioning and pseudoconditioning. A robust CS-elicited neuronal response persisted throughout the first 2 days of trace conditioning, declining gradually across subsequent training sessions. In contrast, the magnitude of the CS-elicited excitatory response during pseudoconditioning began to decline within the first 10 trials. Neurons exhibiting excitatory responses to the CS during trace conditioning also exhibited excitatory responses to the US that were significantly greater in magnitude than US-elicited responses during pseudoconditioning. CS-elicited decreases in neuronal activity became more robust over the course of trace conditioning compared to pseudoconditioning. Reductions in activity during the CS interval consistently preceded excitation in both training groups, suggesting that the CS-elicited decreases in neuronal activity may serve to increase the signal-to-noise ratio of the excitatory response to the tone. Taken together, these data suggest that the caudal AC is involved early in trace eyeblink conditioning and that maintenance of the CS-elicited excitatory response may serve to signal the salience of the tone.