Human follicular fluid collected by laparoscopic oocyte pick-up during IVF was studied with a computer-assisted semen analyser to evaluate the effect of hFF on human sperm motility and velocity. Freshly ejaculated human sperm were washed with phosphate buffered saline and mixed with either PBS or hFF. At various incubation periods of time, hFF increased both sperm motility and velocity as compared with control (P less than 0.01). After incubation of sperm with hFF at 37 degrees C and 5% CO2 in air for 0, 1, 3, 6, and 12 h, the amplitude increase of motility were 49%, 77%, 330%, 2020%, and 3340% when individual control motility was considered to be 100%. The amplitude increase of curvilinear velocity were 43%, 51%, 67%, 152%, 278%, respectively. Comparison of the motility and velocity of the sperm treated with hFF between 0 and 12 h, showed that hFF preserved both motility and velocity in vitro (P less than 0.01). The stimulatory effect of hFF was retained after boiling at 100 degrees C for 30 min, or after being filtered through Amicon membrane cones, but it disappeared if the hFF had been pre-treated with chymotrypsin. However, hFF did not stimulate the motility and velocity of unwashed sperm in freshly ejaculated human semen. A non-dialyzable and heat-stable factor(s) with a molecular weight below 50,000 in hFF may improve and maintain the motility and velocity of washed human sperm. Whether this factor could be used to improve pregnancy rate in assisted reproduction awaits further investigation.