Seminal plasma constituents were separated on Sephadex G200 gel columns. The column eluate was analysed with regard to protein content, ATPase activity and promotive activity on sperm progressive motility. Two different chromatographic fractions were also subjected to electron microscopy after sedimentation by preparative ultracentrifugation. A maximum promotive value on sperm progressive motility coincided with a maximum ATPase activity value in a single peak from seminal plasma eluted first on the column and containing less protein than the other peaks appearing later in the chromatogram. This first peak was the only one containing ATPase activity and membrane-surrounded organelles named prostasomes. Other peaks, rich in protein but lacking ATPase and prostasomes, displayed a moderate and rather irregular pattern in reference to promotive activity on sperm progressive motility. Evidence is given that the positive effect by prostasomes is specific on sperm progressive motility. Hence, procedures aiming at a change of membrane integrity of the prostasomes resulted in diminished effects on sperm progressive motility. This could be explained by a probable dissipation of the electrochemical gradient of calcium ions.