This study evaluated the effect of ovum aging on the in vitro fertilizability of mouse ova. Over 1347 ova were evaluated. Serial trypsin digestion of in vitro and in vivo aged ova revealed an increase in zona digestion time (0.25% trypsin) beginning at 40 hr, which increased over a 40-hr period and resulted in the unfertilized zona becoming as "hard" as the fertilized embryo zona. In vitro fertilizability showed a rapid decrease as zona hardening occurred with loss of cortical granules as assessed by electron microscopy. These data suggest that the window of fertilizability is "closed" by a spontaneous zona reaction occurring at about 55 hr post-human chorionic gonadotropin with loss of cortical granules and zona hardening as manifested by increasing zona digestion time with 0.25% trypsin.