The effects of the time of ova recovery following hCG injection, the presence of cumulus cells, duration of sperm preincubation time, and pH on in vitro fertilization in C57BL/6 mice were investigated. Significantly more ova were recovered at 14 h than at 12 h post-hCG injection. Although the number of ova recovered at 16 h was similar to that at 14 h, the percentage of ova showing degeneration increased. The presence or absence of cumulus cells had no effect on ovum fertilization rates, although sperm incubated with cumulus-intact ova underwent the acrosome reaction sooner than those incubated with ova lacking cumulus cells. Sperm motility was sustained slightly longer in the presence of cumulus-free ova than in the presence of cumulus-intact ova. The average percent fertilization of eggs combined with sperm preincubated 1 h was higher than that of sperm preincubated 0 and 0.5 h. Longer preincubation times resulted in a linear decrease in the percent motility and an increase in the percent acrosome reactions. A plot of the number of sperm attaching to the egg vs. coincubation time produced a bell-shaped curve in each case. The greatest number of sperm attaching to the egg occurred between 45 min and 1 h. When the medium was at pH 7.4, fertilization rates were higher than at pH 7.0, 7.2, or 7.6, as were the percent sperm motility and the number of sperm attached to ova. A pH of 7.6 induced 60% of the sperm to undergo the acrosome reaction immediately, and within 1 h all motility was lost.