Background and Objectives: The gap prepulse inhibition of the acoustic startle response has been used to screen tinnitus in an animal model. Here, we examined changes in the auditory late response under various conditions of gap prepulse inhibition.Subjects and Methods: We recruited 19 healthy adults (5 males, 14 females) and their auditory late responses were recorded after various stimuli with or without gap prepulsing. The N1 and P2 responses were selected for analysis. The gap prepulse inhibition was estimated to determine the optimal auditory late response in the gap prepulse paradigm.Results: We found that the gap per se generated a response that was very similar to the response elicited by sound stimuli. This critically affected the gap associated with the maximal inhibition of the stimulus response. Among the various gap-stimulus intervals (GSIs) between the gap and principal stimulus, the GSI of 150 ms maximally inhibited the response. However, after zero padding was used to minimize artifacts after a P2 response to a gap stimulus, the differences among the GSIs disappeared.Conclusions: Overall, the data suggest that both the prepulse inhibition and the gap per se should be considered when using the gap prepulse paradigm to assess tinnitus in humans.