This study was undertaken to detect key parameters of rat sperm motion in relation to male fertility by comparing the differences in sperm motion induced by treatment with alpha-chlorohydrin (ACH), known to produce spermatotoxicity, and nitrobenzene (NTB), known to produce testicular toxicity. Male rats received ACH (5 or 20 mg/kg/day) or NTB (60 mg/kg/day) for either 3 days or 18 days. Epididymal sperm was assessed for motility using a Hamilton-Thorne Sperm Analyzer (HTM-IVOS). Numerical data for statistical analysis and graphical renditions of sperm motion using parameters in radar charts and reconstructed sperm tracks were analyzed to evaluate sperm motion. Males were allowed to copulate with untreated females and cesarean sections were conducted in order to examine the effects of drug administration on male fertility. Linearity of sperm track (linearity (LIN) and/or straightness (STR)) decreased and/or beat cross frequency (BCF) increased only in ACH groups (5 or 20 mg/kg/day), although the percentage of motile sperm, sperm velocities (average path velocity (VAP), curvilinear (VCL), and straight line velocity (VSL)) and amplitude of lateral head displacement (ALH) decreased on Day 18 in both ACH and NTB (60 mg/kg/day) groups. Furthermore, from the individual reconstructed sperm tracks, it was clear that ACH-treated spermatozoa were characterized by abnormal motion ("jerking") with low vigor (low velocities) and little or no forward progression. Finally, only ACH treatment led to a reduction in pregnancy rate or infertility. Therefore, our results suggest that linearity (especially VSL, STR and LIN) in sperm motion is a key parameter for assessing a chemical's potential to induce male infertility.