Background—In the female genital tract, vaginal colposcopy, endometrial mucosal integrity and inflammatory mediators are potential in vivo biomarkers of microbicide and contraceptive safety. Study Design—A randomized, blinded crossover trial of 18 subjects comparing effects of Gynol II (putative inflammatory gel), HEC (putative inert gel) and no gel exposure on endometrial and vaginal epithelial integrity and endometrial and vaginal inflammatory markers (IL-1β, IL-6, IL-8, MCP-1, MIP-1α, MIP-1β, RANTES, TNF-α, IL-1RA, IL-10, SLPI). Results—Gynol II was associated with more vaginal lesions. No endometrial disruptions were observed across conditions. In the vagina, RANTES (p=0.055) and IL-6 (p=0.04) were higher after HEC exposure than at baseline. In the endometrium, IL-1β (p=0.003) and IL-8 (p=0.025) were lower after Gynol II cycles than after no gel. Conclusions—Gynol II and HEC may modulate inflammatory markers in the vagina and endometrium. How these changes relate to infection susceptibility warrants further study.