Objective: To determine if human spermatozoa could be immobilized and intracellular calcium measurements made on individual cells to measure what proportion can respond to P. Design: Spermatozoa were loaded with Fura 2 (Sigma Chemical Co., Poole, Dorset, United Kingdom) and suspended in 10% gelatin at 37°C. A thin layer of the suspension was cooled to room temperature (20°C to 25°C) and [Ca2+](i) was measured with a fluorescence microscope equipped with dual wavelength excitation and an image analysis system. Setting: University-based laboratory. Participants: Semen was obtained from four fertile donors to a donor insemination program. Interventions: None. Main Outcome Measures: [Ca2+](i) was calculated from the ratio of Fura 2 fluorescence excited at 340 nm and that excited at 366 nm. Results: One hundred six of 114 sperm examined (93%) demonstrated a significant response to P but the size and duration of the response was variable. Conclusion: These data demonstrate that most sperm can respond to P.