Spermatozoa from fertile mice heterozygous for tw32, a recessive lethal allele of the T/t locus, were compared to normal spermatozoa in a fertilization in vitro system. The rate of egg penetration following insemination in vitro was determined for epididymal spermatozoa from C57BL/6-tw32 + mice and for epididymal spermatozoa from C57BL/6- + / + mice. At one hour after insemination, the mean of penetration ± standard deviation for spermatozoa from BL/6-tw32 + mice was 20% ± 2.1 (109 eggs observed, 5 experiments), while the mean for spermatozoa from BL/6- +/+ mice was 1% ± 1.5 (107 eggs observed, 4 experiments). By five hours post-insemination, the levels of egg penetration were not significantly different. These results suggest that tw32 increases the initial rate of egg penetration. Preliminary observations of spermmotility and sperm-egg association at one hour post-insemination in vitro do not support the hypothesis that this earlier penetration is due to improved sperm progress to the egg. Rather, the earlier penetration may be a result of changes in the timing of capacitation, the acrosome reaction, or sperm-egg fusion. It is possible that the earlier penetration may play a role in the distortion of the transmission ratio of tw32.