Evidence from brain imaging studies has indicated involvement of the prefrontal cortex (PFC) in time perception; however, the role of this area remains unclear. To address this issue, we recorded single neuronal activity from the PFC of two monkeys while they performed a duration-discrimination task. In the task, two visual cues (a blue or red square) were presented consecutively followed by delay periods and subjects then chose the cue presented for the longer duration. Durations of both cues, order of cue duration [long–short (LS) or short–long (SL)] and order of cue colour (blue–red or red–blue) were randomized on a trial-by-trial basis. We found that subjects responded differently between LS and SL trials and that most prefrontal neurones showed significantly different activity during either the first or the second delay period when comparing activity in LS and SL trials. The present result offers new insights into neural mechanisms of time perception. It appears that, during the delay periods, the PFC contributes to implement a strategic process in temporal processing associated with a trial type (LS or SL) such as representation of the trial type, retention of cue information and anticipation of the forthcoming cue.