The motility pattern of mammalian spermatozoa changes during migration in the female genital tract and during incubation in vitro. This change in motility is termed hyperactivation. Hyperactivated spermatozoa swim vigorously in 'whiplash', 'figure-8' or 'small circle' trajectories. In this study, a quantitative analysis was carried out of the changes in the motility pattern of hamster spermatozoa during incubation to investigate the mechanism regulating hyperactivation. In the culture system used in this study, hyperactivation occurred 4 h after incubation. Several parameters in the analysis of sperm movement pattern were examined. Curvilinear velocity, average path velocity and straightness abruptly increased between 2 and 4 h. However, linearity, amplitude of lateral head displacement, beat cross frequency and average wavelength gradually changed with time. In the analysis of flagellar bending, the bend angles were measured after dividing images of the flagellum into short lengths. Flagellar bending changed in different manners in each region during incubation. The asymmetry in the direction of the curve of the head gradually increased with time in the first half of the flagellum. The flexibility, which was determined using the amplitude of bending and the rate of change in bend angles, abruptly decreased between 10 min and 1 h, and then increased between 2 and 4 h in the first half of flagellum. These results indicate that complex physiological changes occur before hyperactivation.