Temporal order memory was analyzed using a spontaneous object recognition (SOR) paradigm in rats. In SOR, animals were allowed to explore freely two or five different objects sequentially in the sample phase, and then, two different objects shown in the sample phase were simultaneously presented, and exploration time of object shown earlier compared with that shown later was regarded as a discrimination index. Here we investigated the effects of (1) number of sample items, (2) sample exposure interval, and (3) retention time between the sample and test phases on temporal order memory in SOR. In experiment 1, rats showed significant discrimination between the objects presented earlier and later under five-item, but not two-item, condition. In experiment 2, using the five-item condition, longer retention time (between sample and test phases) induced lower discrimination and longer sample exposure interval produced higher discrimination. In experiment 3, using the two-item condition, longer sample exposure interval produced higher discrimination and longer retention time tended to make rats' discrimination worse. Taken together, these results indicate that rats' performance in this test paradigm was better with more sample items and longer exposure intervals as well as shorter retention time, suggesting that the familiarity to items and the temporal distance from the present to the occurrence of events affect temporal order memory.