Background: Small intensive pharmacokinetic (PK) studies of medications in early-phase trials cannot identify the range of factors that influence drug exposure in heterogenous populations. We performed PK studies in large numbers of HIV-infected women on nonnucleoside reverse transcriptase inhibitors (NNRTIs) under conditions of actual use to assess patient characteristics that influence exposure and evaluated the relationship between exposure and response. Methods: Two hundred twenty-five women on NNRTI-based antiretroviral regimens from the Women's Interagency HIV Study were enrolled into 12-hour or 24-hour PK studies. Extensive demographic, laboratory, and medication covariate data were collected before and during the visit to be used in multivariate models. Total NNRTI drug exposure was estimated by area under the concentration-time curves. Results: Hepatic inflammation and renal insufficiency were independently associated with increased nevirapine exposure in multivariate analyses; crack cocaine, high fat diets, and amenorrhea were associated with decreased levels (n = 106). Higher efavirenz exposure was seen with increased transaminase, albumin levels, and orange juice consumption; tenofovir use, increased weight, being African American, and amenorrhea were associated with decreased exposure (n = 119). With every 10-fold increase in nevirapine or efavirenz exposure, participants were 3.3 and 3.6 times as likely to exhibit virologic suppression, respectively. Patients with higher drug exposure were also more likely to report side effects on therapy. Conclusions: Our study identifies and quantitates previously unrecognized factors modifying NNRTI exposure in the real-world setting. Comprehensive PK studies in representative populations are feasible and may ultimatley lead to dose optimization strategies in patients at risk for failure or adverse events.