To optimize the Hamilton-Thorn Motility Analyzer (HTM; Hamilton-Thorn Research, Beverly, MA) for use in reproductive toxicology studies with rat spermatozoa, the accuracy and precision of the instrument were assessed under a variety of instrument settings. Videotapes of both fast- and slow-swimming sperm were analyzed repeatedly to obtain data across a range of sperm velocities as might be encountered as a consequence of exposure to reproductive toxicants. Acquisition rates were varied across the HTM menu choices (30, 19, 10, or 7 frames/sec) as were the number of frames analyzed (5 to 20) at each framing rate. For fast-swimming samples (mean straight-line velocity (VSL) approximately 130 microns/sec) generally good agreement between computer-assisted sperm analysis (CASA) and manually obtained data was found for percentage of motile sperm and straight-line velocity; i.e., CASA values were within 10% of manual values for most frame/rate combinations. The accuracy of these measures held true over a wide range of sperm concentrations and percentage motilities. However, CASA measures were less accurate for sperm samples of lower velocities (mean VSL approximately 50 microns/sec and mean VSL approximately 30 microns/sec) in that the velocity of very slow sperm was overestimated (particularly at 30 frames/sec). A soft-ware change (6.5R) and performing analyses at 19 instead of 30 frames/sec improved straight-line accuracy for the slow sperm and enhanced the discrimination between fast (presumably control) and slow (presumably treated) sperm samples. These data show that this motility analyzer could be successfully configured to evaluate rodent sperm samples. The use of such CASA systems in toxicology studies will provide valuable information that may improve human reproductive risk assessment.