The present study intended to develop the hypoosmotic swelling (HOS) test and to determine an appropriate hypoosmotic solution (HS) for its use in epididymal spermatozoa from ram, thus allowing the evaluation of the membrane functional integrity. We also tested if a 15. min exposure time could generate the same results as longer incubation times (30, 45 and 60. min). Ten different hypoosmotic solutions (50, 75, 100, 125, 150, 175, 200, 250, 275, and 300. mOsm/kg) were prepared from sodium citrate and fructose diluted in bidistilled water and were employed at 37. °C. According to the percentage of reacted spermatozoa, 75-100. mOsm/kg solutions were the most sensitive (P<0.05, n=15). Also, the different exposure times did not show any significant effect (P>0.05, n=15); therefore allowing 15. min to be considered sufficient incubation time. © 2013 Elsevier B.V.