Abstract To investigate whether prefrontal neurons temporarily retain information regarding multiple spatial positions, single-neuron activity was recorded while monkeys performed a delayed sequential reaching task, in which they needed to remember two cue positions out of three and their temporal order of presentation. Most neurons were also tested on a conventional delayed reaching task, in which they needed to remember one cue position during the delay. Among 72 neurons that exhibited significant delay-period activity, one group of neurons ( n = 19) exhibited delay-period activity only when a visual cue was presented at one of the three positions (position-dependent). Of these, 6 neurons exhibited this activity when a cue was presented at that position independent of the temporal order, whereas 13 neurons exhibited this activity only when a cue was presented at that position in a particular temporal order (e.g., as the first cue or the second cue). Another group of neurons ( n = 39) exhibited delay-period activity only when visual cues were presented at two positions out of three (pair-dependent). Of these, 7 neurons exhibited pair-dependent activity independent of the temporal order of cue presentation. However, 32 neurons exhibited this activity only when two cues were presented in a particular temporal order. The remaining 11 neurons exhibited non-differential activity during the delay period and 3 neurons exhibited miscellaneous activity. These results show that a single prefrontal neuron can retain information regarding two spatial positions, and that, to retain two spatial positions and the temporal order of cue presentation, new types of delay-period activity emerged; i.e., pair-dependent activity and temporal order-dependent activity. Both types of activity could be a mechanism for simultaneously retaining two items of spatial information and for effectively combining multiple spatial information by a single neuron. In addition, the presence of delay-period activity with position-dependency, pair-dependency and temporal order-dependency suggests that the dorsolateral prefrontal cortex plays an important role in planning sequential behaviors.