This study aimed to assess whether a single oral dose of the nonnucleoside reverse transcriptase inhibitor efavirenz can alter CYP3A in vivo. In 12 healthy participants individual CYP3A activity was quantified using a semisimultaneous methodology (midazolam orally and 6 hours later intravenously) both alone and during a period of 22 days after a single oral dose of 400 mg efavirenz. Twelve hours after efavirenz administration, midazolam apparent oral clearance was significantly increased by 70%, and midazolam systemic clearance after intravenous administration was significantly increased by 27%. Similar effects were still present on day 6, after which midazolam clearances slowly returned to baseline on day 22. At least on day 1, the midazolam clearance increase is consistent with the in vitro observed CYP3A activation. The onset of an efavirenz treatment will almost immediately result in enhanced elimination of CYP3A substrates.