To investigate the influence of formular composition on the in vitro and in vivo properties of poloxamer-based thermosensitive in situ hydrogel for vaginal administration with nonoxinol as the model drug.Two nonoxinol-containing in situ hydrogel formulations, GEL 1 (18% poloxamer 407/1% poloxamer 188) and GEL 2 (18% poloxamer 407/6% poloxamer 188) were compared with respect to the rheological property, in vitro hydrogel erosion and drug release, as well as vaginal residence in different animal models, including drug quantitation of vaginal rinsing in mice and 99mTc isotope tracing in rats.GEL 1 and GEL 2 exhibited similar rheological properties and phase transition temperatures ranged from 28 to 30℃.Diversity (5%) in the contents of poloxamer 188 between GEL 1 and GEL 2 resulted slight (but not significant) difference in the rates of both hydrogel erosion and drug release.When compared in vivo, GEL 1 differed from GEL 2 obviously in rats in the intravaginal residence (10 h for GEL 1 versus 7 h for GEL 2), while which was difficult to be distinguished in mice.The content of poloxamer 188 influenced hydrogel erosion, drug release and intravaginal residence of poloxamer-based thermosensitive in situ hydrogel.For in vivo evaluation of intravaginal clearance, rats may be a more suitable animal model than mice.