A method for preparing stored unfrozen stallion spermatozoa for the zona-free hamster oocyte penetration test (HOPT) and a subsequent comparison of fresh and stored sperm by the HOPT were evaluated. In Experiment 1, sperm from 4 stallion ejaculates, cooled to 4 degrees C and stored for 24 h, were treated with 60, 90 and 120 microM of dilauroylphosphatidyl-choline (PC12) liposomes to initiate the acrosome reaction. The percentage of motile and acrosome-reacted (AR) sperm were recorded after 8, 15 and 30 min of incubation at 39 degrees C, by automated image analysis. Liposome concentration did not affect motility during 8- or 15-min incubations. Sperm samples treated with 120 microM PC12 had fewer (P less than 0.05) motile sperm after 30 min and had a higher (P less than 0.05) percentage of AR sperm at all times than did samples treated with 60 microM PC12. In Experiment 2, sperm cooled and stored for 0, 24 and 72 h from 5 stallion ejaculates were treated with 120 microM PC12 for 8 min and incubated with frozen-thawed, zona-free hamster oocytes. There was no difference (P greater than 0.05) in the percentage of eggs penetrated by sperm stored for 0, 24 and 72 h (77, 80 and 75%) but the average number of penetrating sperm per penetrated egg was lower (P less than 0.01) after 72 h of storage (5.9 and 6.1 vs 2.9). Results of this study indicate that stallion sperm can be stored for at least 24 h at 4 degrees C without change in sperm characteristics measured here, and the HOPT test may be useful in indicating a decline in fertilizing potential with prolonged storage.