Contents: The present study intended to develop the hypoosmotic swelling (HOS) test in alpaca for its use in epididymal spermatozoa, to evaluate the membrane functional integrity and determine an appropriate hypoosmotic solution and whether the incubation time of 15 or 60min is sufficient for the execution of the test. Hypoosmotic solutions (HS) with the following concentrations were used: 50, 100, 150, 200 and 275mOsm/kg of sodium citrate tribasic dihydrate and d-fructose. Ten microlitres of epididymal sperm sample was mixed in 150μL of the respective HS and incubated for 15 or 60min at 38°C. From the proportion of reacted (swollen) spermatozoa, the 150mOsm/kg HS was the most sensitive (p<0.05). The exposure times (15 and 60min) did not have significant differences (p>0.05) in the proportion of both strong- and total-coiled sperm tails. In conclusion, 150mOsm/kg HS and 15min exposure time are optimal to evaluate the plasma membrane functional integrity through the HOS test in alpaca epididymal spermatozoa. © 2012 Blackwell Verlag GmbH.