Three ejaculates from each of 3 stallions were used to evaluate a computerized system (Hamilton-Thorn motility analyzer; HTMA) for measuring equine spermatozoal motility. Variance components (ejaculate-within-stallion, chamber-within-ejaculate, and microscopic field-within-chamber) were determined for each stallion after diluting ejaculates to 25 X 10(6) spermatozoa/ml with a skim milk-glucose seminal extender. The HTMA was compared with frame-by-frame playback videomicrography (VIDEO) for determining: percentage of spermatozoal motility and spermatozoal number in microscopic fields; curvilinear velocity and straight-line velocity of individual spermatozoa for 5 track types; and repeatability of those velocity measurements. The effect of spermatozoal number per microscopic field on incidence of intersecting spermatozoa and the outcome of intersecting spermatozoa also were evaluated. Greatest variability in motility measures was generally attributed to the microscopic field-within-chamber component. The HTMA was highly correlated with VIDEO for estimation of spermatozoal numbers per microscopic field (r = 0.99; P <0.001) and motility (r = 0.97; P <0.001); however over the entire range of spermatozoal numbers, the HTMA yielded higher spermatozoal numbers per microscopic field (P <0.05) and higher motility (P <0.05) than did VIDEO. The HTMA- and VIDEO-derived measurements of curvilinear and straight-line velocities were highly correlated for all spermatozoal track types, but both measures were higher (P <0.05) by use of the HTMA than by use of VIDEO for most track types. For 3 of 5 track types, measurements of curvilinear and straight-line velocities were less variable (P <0.05), using the HTMA, rather than VIDEO. Using the HTMA, the number of intersecting spermatozoa was highly correlated with spermatozoal numbers per microscopic field (r = 0.97; P <0.001). The percentage of erroneous track interpretations involving intersecting spermatozoa was high (85.3 +/- 2.7%). The HTMA was a reliable system for determining percentage of spermatozoal motility and velocity measures in video recordings of equine semen diluted to spermatozoal concentration of 25 X 10(6)/ml prior to evaluation.