Experiments were conducted to determine some of the biological variables that may affect sperm penetration of human cervical mucus in vitro. Quantitative tests of cervical mucus penetration were carried out to determine the percentage of successful collisons (PSC) between seminal spermatozoa and the semen‐mucus interface. Fifteen duplicate comparisons and 15 triplicate comparisons of PSC values were made, each using individual samples of semen and mucus. In most cases the difference between any two comparisons was less than 10%, and there was no correlation between the magnitude of the difference between tests and the absolute value of the PSC. The triplicate comparison showed no correlation between the PSC values and the location of the mucus in the collection catheter (proximal, middle, distal). In 15 experiments the semen was serially diluted with an aliquot of its own plasma to determine the effect of sperm concentration on the PSC. No effects were observed until the sperm concentration fell below 10 × 106 sperm/ml, when the PSC appeared to increase. These results indicate that the tests should be applicable to all but the most severely oligospermic semen samples. Copyright © 1984 Alan R. Liss, Inc.