Description: Background.  Microbicide toxicity may reduce the efficacy of topical preexposure prophylaxis for human immunodeficiency virus (HIV) transmission. Noninvasive quantitative measures of microbicide toxicity would usefully inform microbicide development. Methods.  Ten subjects received 3 one-time interventions: 5 mL of Normosol-R fluid alone (negative control), 5 mL of 2% nonoxynol-9 (N-9) gel, and 5 mL of Normosol-R with coital simulation and sigmoidoscopic biopsy (CS + BX). Each dose of N-9 and Normosol-R contained 500 µCi of 99m technetium–diethylene triamine pentaacetic acid. Plasma and urine radioactivity was assessed over 24 hours. Results.  The plasma radioisotope concentration peaked 1 hour after N-9 dosing. The mean maximum radioisotope concentration after N-9 receipt was 12.0 times (95% confidence interval [CI], 6.8–21.0) and 8.4 times (95% CI, 5.2–13.5) the mean concentration after Normosol-R control receipt and CS + BX receipt, respectively; paired differences persisted for 24 hours. After N-9 dosing, the urine isotope level was 3.6 times (95% CI, 1.1–11.4) the level observed 8 hours after Normosol-R control receipt and 4.0 times (95% CI, 1.4–11.4) the level observed 4 hours after CS + BX receipt. Permeability after CS + BX receipt was greater than that after Normosol-R control receipt in 0–2-hour urine specimens only (mean permeability, 2.4; 95% CI, 1.0–5.8) but was not greater in blood. Conclusions.  Plasma sampling after rectal radioisotope administration provided quantitative estimates of altered mucosal permeability after chemical and mechanical stresses. Permeability testing may provide a useful noninvasive adjunct to assess the mucosal effects of candidate microbicides. Clinical Trials Registration.  NCT00389311.