Two morphologically distinct auditory middle latency response (MLR) wave forms can be recorded from the surface of the guinea pig brain. The temporal response is recorded from the temporal lobe contralateral to the stimulus ear, and the midline response is recorded over the posterior midline. Experimental evidence suggests that different neural generators contribute to the two responses. Furthermore, it appears that the temporal response principally reflects activity of the primary auditory pathway while the midline response reflects non-primary pathways. Although it is known that neurons throughout the auditory pathway exhibit distinct binaural interaction (BI) properties, thus far there have been no systematic attempts to differentiate the MLR wave forms in response to binaural stimulation. The purpose of this study was to determine if binaural click stimulation could functionally differentiate the midline and temporal MLR responses in the guinea pig. Binaural click stimulation caused a significant decrease in temporal MLR peak amplitudes, and a significant increase in midline MLR amplitudes. The fact that different BI patterns were observed suggests that the two MLR components are functionally distinct. The data further support the hypothesis that the midline and temporal MLR in guinea pigs reflect different neural generators and pathways.