Sperm motion analysis following exposure to a reproductive toxicant is one means of evaluating the functional integrity of the testis and epididymis. In this study we sought to determine whether the method used to collect sperm from the proximal cauda epididymidis, where sperm are not completely mature, has a significant influence on sperm motion parameters. Two methods of collecting rat sperm for motion analysis were used: one based on an aspiration technique selected from the literature; the other, a new approach based on diffusion of sperm from the epididymal tubule. The two methods were tested for sensitivity to effects on sperm motility parameters 4 days after a single exposure to ethane dimethanesulphonate (EDS). Since EDS is known to decrease serum testosterone (T), an additional group of rats received T-filled implants just prior to dosing to determine if the decrease in serum T alone had an effect on sperm motility. The results of the study yielded strikingly different interpretations of the effect of a 65 mg/kg BW dose of EDS on the motility of sperm taken from the proximal cauda epididymidis. Sperm collected by "aspiration" showed no significant decrease in the percentage of motile or progressively motile sperm compared to vehicle-treated animals. On the contrary, sperm collected by "diffusion" showed large, significant decreases in the percentages of both motile and progressively motile sperm. This difference was due largely to lower percentages of motile and progressively motile sperm in control sperm samples collected by aspiration. Similarly, the motion parameters of sperm collected by the aspiration method were unaffected by EDS/T treatment.(ABSTRACT TRUNCATED AT 250 WORDS)