Computer-assisted motion analyses (CASA) and flow cytometry were used to evaluate stallion spermatozoa prior to and after cryopreservation. Spermatozoa were pretreated with: (1) Hepes-buffered medium (SHB); (2) phosphatidylserine (PS) liposomes; or (3) liposomes composed of both PS and cholesterol (PSCH) prior to dilution in either SHB or skim milk-egg yolk extender (SMEY). After cooling to 5 degrees C in SHB, PS and PSCH pretreatment (23%). Spermatozoal motion parameters were higher for spermatozoa diluted in SMEY than dilution in SHB. In Experiment 2, motion parameters were compared for spermatozoa pretreated with PSCH liposomes and cryopreserved in either SMEY or a high salt-skim milk-egg yolk extender (CO). Spermatozoal motion characteristics were similar for all spermatozoal treatments after cooling at 5 degrees C. After cryopreservation, PSCH liposome-treated samples had higher percentages of motile spermatozoa than untreated samples regardless of freezing extender. Samples frozen in CO medium had higher percentages of motile spermatozoa than samples frozen in SMEY (P < 0.05; 63% in CO + PSCH and 54% in CO vs 55% in SMEY + PSCH and 48% in SMEY, respectively). In Experiment 3, spermatozoa were treated with dilauroylphosphatidylcholine (PC12) to induce the acrosome reaction. The percentages of viable cells and viable acrosome-reacted spermatozoa were higher for fresh spermatozoa than for cryopreserved spermatozoa (P < 0.05), but were not affected by PSCH liposome treatment (P > 0.05). Addition of PSCH liposomes improved recovery of motile spermatozoa after cryopreservation but did not affect the ability of spermatozoa to undergo a PC12-induced acrosome reaction.