To investigate the involvement of flotillin-1 in acute experimental testicular torsion, we examined the expression and cellular localization of flotillin-1 and cathepsin D in the rat testis with ischemia/reperfusion (I/R) injury. Western blot analysis showed that the expression of flotillin-1 increased significantly 6 h after I/R and that the level remained elevated for 48 h. Immunohistochemically, flotillin-1 was constitutively localized in some Sertoli cells, peritubular myoid cells, and interstitial cells in the normal testis. After I/R injury, Sertoli cells in the damaged tubules were intensely immunostained for flotillin-1 at 24 and 48 h after I/R. Flotillin-1 was also detected in some inflammatory cells in the interstitial space around damaged tubules. Furthermore, flotillin-1 was colocalized with cathepsin D, a lysosomal marker, in normal testis (mainly in Sertoli cells), and the colocalization was greater in Sertoli cells and macrophages in I/R injured testes. Therefore, we postulate that flotillin-1 immunoreactivity is increased in some Sertoli and inflammatory cells (especially in ED1-positive activated macrophages) in testicular torsion and that flotillin-1 in the injured testis associates with lysosomes in Sertoli cells and macrophages, activating subsequent signals in inflammatory macrophages and Sertoli cells after I/R.