ABSTRACT: Ejaculated mammalian spermatozoa acquire competence to fertilize oocytes by a two-step process: capacitation followed by acrosome reaction. The biochemical and biophysical modifications occurring in vivo in the female reproductive tract can be reproduced in vitro, and previous studies have suggested a capacitative role for adenosine A1 receptor (A1R). Mice with a targeted disruption of the Adora 1 gene (A1R–/– mice) provide a useful model for better understanding the role of the A1R in fertility. Murine spermatozoa express A1R in the head, neck, midpiece region, and tail. The number of capacitated spermatozoa incubated in human tubal fluid was significantly reduced in A1R–/– compared with A1R+/+ and A1R+/– spermatozoa. The difference between A1 R+/+ and A1R−/− mouse spermatozoa was mainly in the time necessary to reach the maximum percentage of capacitation. A1R+/+ murine sperm obtained the full state of capacitation within 90 minutes whereas A1R−/− sperm required 240 minutes. Caffeine, a known antagonist of A1 and A2A adenosine receptors, lowered the number of capacitated sperm and affected the time of capacitation in a dose-dependent manner, mimicking the effects of the lack of A1 receptors. Although number, motility, and viability of A1R−/− murine sperm was not significantly different from A1R+/+ mouse spermatozoa, a significant reduction of the number of pups produced by A1R−/− male mice suggests that A1 receptors must be fully operative to accomplish the optimal degree of capacitation and thereby fertilization.