Motion characteristics of cooled stallion spermatozoa in 2 freezing extenders were studied. Ejaculates from 8 stallions were split into treatments and cooled in thermoelectric cooling units at each of 2 rates. Cooling started at 37 degrees C for Experiments 1 and 3 and at 23 degrees C for Experiments 2 and 4, at a rate of -0.7 degrees C/min to 20 degrees C and from 20 to 5 degrees C, at either -0.05 degrees C/min (Rate I) or -0.5 degrees C/min (Rate II). Percentages of motile (MOT) and progressively motile spermatozoa (PMOT) were determined at 6, 24 and 48 h. Treatments in Experiment 1 were modified skim milk extender (SM); SM + 4% egg yolk (EY); SM + 4% glycerol (GL); and SM + 4% egg yolk + 4% glycerol (EY + GL). At 24 and 48 h, MOT and PMOT were lowest (P < 0.05) for spermatozoa extended in SM + EY; spermatozoa in SM + GL had the highest MOT and PMOT. Thus, glycerol partially protected spermatozoa against the effects of cooling after long-term storage. Treatments in Experiment 2 were SM, semen centrifuged and pellet resuspended in SM (SMc), SM + EY, and semen centrifuged and pellet resuspended in SM + EY (EYc). Spermatozoa in SM + EYc had the highest (P < 0.05) PMOT at 24 h and MOT and PMOT at 48 hours. Spermatozoa in SM + EY (not centrifuged) had the lowest MOT and PMOT at 24 and 48 h, respectively. There was a detrimental interaction between egg yolk and seminal plasma. Extenders in Experiment 3 were Colorado extender (CO3), CO3 + 4% egg yolk (EY), CO3 + 4% glycerol (GL), and CO3 + 4% egg yolk + 4% glycerol (EY + GL). Spermatozoa in CO3 + EY had the lowest (P < 0.05) PMOT at 24 and 48 h. CO3 did not protect spermatozoa cooled in the presence of seminal plasma. Therefore, in Experiment 4 we tested CO3 with seminal plasma present (control) and semen centrifuged and pellet resuspended in CO3 (CO3c), CO3 + EY (EYc), CO3 + GL (GLc) and CO3 + EY + GL (EY + GLc). Spermatozoa in CO3 had the lowest (P < 0.05) MOT and PMOT at all time periods, which suggested a detrimental interaction of this extender with seminal plasma.