The clinical use of the hypoosmotic swelling test (HOST) to identify spermatozoa with a functional intact membrane has been reported for humans and domestic species, including the dog. Currently, it is recommended that canine spermatozoa be incubated with the hypoosmotic solution for periods that range from 30 to 60 min. In an attempt to simplify the test, it was hypothesized that the degree of the hypoosmotic response at 1 min of incubation would not be different from the response documented at 60 min after incubation in the hypoosmotic solution at 37 degrees C. The hypoosmotic response of spermatozoa from 50 fresh and 16 frozen-thawed semen samples obtained from 22 adult dogs was recorded at 1 and 60 min of incubation. There were no significant differences between the hypoosmotic response recorded at 1 and 60 min for all evaluated semen samples (P>0.10). The hypoosmotic response recorded for canine spermatozoa from fresh semen samples were greater than that recorded for spermatozoa from frozen-thawed semen, both at 1 min (86.2% compared with 65.2%; P<0.001) and 60 min (85.6% compared with 61.8%; P<0.001). Based on the results of this study, it is recommended to decrease the incubation time of the HOST for canine spermatozoa to as short a period as 1 min. This incubation time should encourage the application of this relatively simple and inexpensive test of canine sperm membrane function in a clinical setting.