Antisera against the circadian clock proteins Period (PER) and Timeless (TIM) were used to construct a detailed time course of PER and TIM expression and subcellular localization in a subset of the ventrolateral neurons (vLNs) in the Drosophila accessory medulla (AMe). These neurons, which express pigment-dispersing factor, play a central role in the control of behavioral rhythms. The data revealed several unexpected features of the circadian clock in Drosophila. First, TIM but not PER was restricted to the cytoplasm of vLNs throughout most of the early night. Second, the timing of TIM and PER nuclear accumulation was substantially different. Third, the two subsets of vLNs, the large and small vLNs, had a similar timing of PER nuclear accumulation but differed by 3-4 hr in the phase of TIM nuclear accumulation. These aspects of PER and TIM expression were not predicted by the current mechanistic model of the circadian clock in Drosophila and are inconsistent with the hypothesis that PER and TIM function as obligate heterodimers. The differing profiles of TIM and PER nuclear accumulation suggest that PER and TIM have distinct functions in the nuclei of vLNs.