HepG2 cell lines that constitutively and stably express human CYP3A4 were constructed in order to study enzyme interactions with CYP3A4 as the only P450 present. CYP3A4 activity and content were assessed by the metabolism of fentanyl, a CYP3A substrate, and Western blots. Northern blots were used to examine the effects of acetaminophen (APAP) on CYP3A4-mRNA. The HepG2 cell lines' CYP3A4 activity was stable over time. High concentrations of APAP inhibited CYP3A4 activity. At lower concentrations, APAP produced a dose-dependent increase in CYP3A4 activity and content. No increases in CYP3A4-mRNA were seen. Incubation with cycloheximide caused a decrease in fentanyl metabolism secondary to a decrease in P450 levels that was prevented by the coincubation with APAP. Additionally, human microsomal CYP3A4 was stabilized by APAP against cytosol-mediated degradation. In our models, APAP appears to increase CYP3A4 activity. This increase appears to be via substrate stabilization. This is the first report that APAP can increase CYP3A4 activity and content in transfected HepG2 cells.