Mammalian sperm hyperactivation (HA) is a change in motility that accompanies capacitation (CAP) and is dependent on calcium (Ca) (Yanagimachi and Usui, Exp Cell Res 89:161, 1974). HA may be important for transport through the female tract and/or for fertilization. To develop an objective and quantitative assay for HA in individual mouse sperm, a computer-assisted motion-analysis system was used to describe sperm translational movements. To determine which movements were characteristic of HA, Ca-dependent motility was identified. This was done by incubating sperm with or without calcium (Ca+ or Ca- sperm, respectively), and determining the range of values for each motility parameter that was present only among Ca+ sperm. To do this, we compared frequency distributions of motility parameter values at the time of maximal CAP (90 min). CAP was monitored by measuring the level of in vitro fertilization and by evaluating the pattern of chlortetracycline binding to individual sperm heads [Ward and Storey, Dev Biol 104:287, 1984]. Two Ca-dependent motility subgroups were apparent: 1) a "slow-speed" subgroup with a curvilinear velocity (Vc) less than 169 microns/sec that had none of the characteristics expected of HA sperm; and 2) a subgroup with higher speeds (Vc greater than 169 microns/sec) and wider-amplitude head movements as measured by curvilinear progressiveness ratio (PRc less than 0.56). The latter subgroup was selected as HA, since the frequencies and time course were similar to those for CAP in the same population. Two media components known to be important for CAP, bicarbonate and bovine serum albumin (BSA) were then tested to determine whether they were necessary for HA. Incubation of sperm without bicarbonate prevented HA, but omitting BSA did not affect HA during the first 3 hrs. These data suggest that HA is not tightly coupled with CAP.