Substance use disorder is associated with disruptions in circadian rhythms. The circadian transcription factor neuronal PAS domain protein 2 (NPAS2) is enriched in reward-related brain regions and regulates reward, but its role in substance use is unclear. To examine the role of NPAS2 in drug taking, we measured intravenous cocaine self-administration (acquisition, dose-response, progressive ratio, extinction, cue-induced reinstatement) in wild-type (WT) and Npas2 mutant mice at different times of day. In the light (inactive) phase, cocaine reinforcement was increased in all Npas2 mutants, while self-administration and motivation were affected sex-dependently. These sex differences were amplified during the dark (active) phase with Npas2 mutation increasing self-administration, reinforcement, motivation, extinction responding and reinstatement in females, but only reinforcement in males. To determine whether circulating hormones are driving these sex differences, we ovariectomized WT and Npas2 mutant femal...