Using a classical conditioning procedure in urethane-anesthetized rats, a light applied to one eye (CS+) was paired with medial forebrain bundle (MFB) stimulation, whereas a light applied to the other eye (CS-) was not paired. Basal forebrain neurons in the substantia innominata, medial globus pallidus, and nucleus basalis magnocellularis responded differentially to CS+ and CS-, with larger responses to CS+. Some neurons were excited by CS+, and others were inhibited. Fifty percent of these neurons responded in the same direction to CS+ and MFB stimulation, and 38% responded in opposite directions. Frontal cortex neurons exhibited similar differential responses; 47% of the differential responses to CS+ were in the same direction as the response to MFB stimulation, and 29% were in the opposite direction. When light to either eye was paired with MFB stimulation, conditioning-related basal forebrain neuron responses of comparable magnitude to left and right eye illumination were observed, providing evidence that association of CS and UCS rather than the eye to which light was applied determined the differential response to CS+. Also, two different intensities of light induced comparable basal forebrain responses when both were paired with the UCS. These experiments provide support for a role of the basal forebrain in conditioning-related neural activity. Furthermore, this preparation can be utilized to investigate transmitter systems that mediate conditioning-related responses of basal forebrain neurons.