The aryl hydrocarbon receptor (AhR) transcription factor is increasingly recognized as functioning in cell cycle control. Several recent reports have shown that AhR activity in the absence of exogenous agonists or presence of the prototypical ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin can affect G1 phase progression in cultured cells. Serum release of serum-starved (G0) 5L rat hepatoma cells triggers transient AhR activation and P4501A1 protein expression concomitant with the G0/G1-to-S phase transition. In contrast, sustained AhR activation in response to TCDD treatment increases p27Kip1 expression in addition to P4501A1, resulting in G1 phase cell cycle arrest. Treating serum-released 5L cells with the alkyne metabolism-based P4501A1 inhibitor 1-(1-propynyl)pyrene results in prolonged AhR activation, enhanced p27Kip1 expression, and G1 phase arrest after serum release. The data are consistent with a cell cycle role for P4501A1 because they show that P4501A1 negatively regulates the duration of AhR action through the metabolic removal of the receptor agonist, thereby preventing AhR-mediated G1 phase arrest.