The ability of human spermatozoa to penetrate zona-free hamster ova was examined following low-temperature capacitation (4 degrees C) in TES-Tris (TEST)-yolk buffer for periods of up to 66 hours. Results obtained from 66 individuals demonstrated that the number of penetrations per ova were increased by an average of 2.5-fold when spermatozoa were capacitated for 42 hours, as compared with 18 hours. Furthermore sperm with extremely poor penetration rates after 18-hour capacitation was often improved by longer capacitation periods. Patients from our infertility clinic with less than 20 X 10(6) spermatozoa/ml of ejaculate had significantly lower penetration rates when compared with patients and donors with greater than 20 X 10(6) spermatozoa/ml (P less than or equal to 0.001). The observed effects of TEST-yolk buffer appear to be related to its ability to preserve sperm motility over prolonged periods, during which increased capacitation of the total sperm population is achieved.