UC781 is a potent nonnucleoside reverse transcriptase inhibitor being investigated as a potential microbicide to prevent transmission of HIV-1 both vaginally and rectally. This study was designed to investigate the in vitro drug release, in vitro permeability/safety, and in vivo pharmacokinetics in rabbits of a vaginal gel prepared with micronized or nonmicronized UC781 (UC781m and UC781nm, respectively). Gels prepared with UC781m had greater in vitro release rates (Franz cells) and permeability/tissue-associated UC781 concentrations than gels prepared with UC781nm (EpiVaginal tissues). Both gels were well tolerated under in vitro conditions compared with controls using EpiVaginal tissues. Following intravaginal administration of both gels to rabbits, tissue concentrations typically ranged from 1,000 to over 10,000 ng/g regardless of dosing regimen (single dose or 7 days once daily dosing) and sampling times (2 and 24 h post-dose). Tissue-associated concentrations were highly variable, and no statistically significant differences were found between test conditions. Plasma levels were generally low after vaginal administration: following a single dose, the concentrations were between 0.5 and 1.0 ng/mL. After 7 days repeated once daily dosing, UC781 concentrations were slightly higher ranging from below 1.0 to about 2 ng/mL, although none of the differences were statistically significant. Based on these results, gels prepared with either form of UC781 led to tissue concentrations well in excess of UC781's EC50 under in vitro conditions (~3 ng/mL).