Objective: To determine whether 12-myristate 13-acetate phorbol ester (PMA) can increase hyperactivated motility of human sperm.Design: A controlled pharmacological study using computer-assisted semen analysis.Setting: Andrology laboratory in a medical research institution.Patient(s): Normal semen was obtained from 48 men.Intervention(s): Washed sperm were exposed to different concentrations of PMA alone or with P and pentoxifylline (PTX) for up to 2 hours and sperm motility measured by a computer-assisted semen analyzer.Main Outcome Measure(s): The percentage of sperm with hyperactivated motility was determined from the motility parameters: curvilinear velocity, linearity, and maximum amplitude of lateral head displacement.Result(s): Phorbol ester PMA increased hyperactivated motility in a dose- and time-dependent manner. At 1 hour, the average increases in hyperactivated motility were as follows: 2 μM, 4.8% ± 1.5%; 6 μM, 9.6% ± 1.5%; and 20 μM, 11.3% ± 2.2%. The PMA effect was not altered when P or PTX were added although each separately had a positive effect on hyperactivation.Conclusion(s): Phorbol ester PMA stimulates human sperm hyperactivated motility, indicating the involvement of protein kinase C in the signal transduction pathway.