AbstractA study of varying combinations of in vitro‐aged sperm and in vivo‐aged ova at 3 hr intervals from 0–24 hr resulted in failures at different steps of the fertilization process during in vitro fertilization of mouse ova. Significant decreases caused by sperm aging, ova aging, and sperm × ova aging interaction were found in sperm penetration. Pronuclear formation was not affected by sperm aging and was enhanced by ova aging, and there was a significant effect of sperm × ova aging interaction. Sperm aging significantly influenced the prometaphase stage of the fertilization process. Therefore, it is suggested that the detrimental fertilization effects resulting from aging gametes are due to different mechanisms in sperm and ova, that these mechanisms are affected at different times, and that they affect different steps in the fertilization