The objective of this study was to determine the effects of oviductal fluid on sperm binding and fertilization in vitro when either bovine oocytes or both spermatozoa and oocytes are incubated in fluid from different regions of the oviduct and at different stages of the oestrous cycle. Oocytes, or oocytes and spermatozoa, were incubated in nonluteal and luteal oviductal fluid obtained from the whole, isthmic or ampullary oviduct of dairy cows. Gametes were washed and coincubated in fertilization medium for 18 h, and then observed for sperm binding and penetration. More spermatozoa bound to oocytes that were preincubated in nonluteal oviductal fluid than to those that were incubated in luteal oviductal fluid; however, there were no differences in fertilization rates between the two treatments. More spermatozoa bound to oocytes incubated in isthmic than in ampullary oviductal fluid. However, if both gametes were incubated in oviductal fluid before their coincubation, binding was higher when both gametes were incubated in ampullary oviductal fluid. By reducing the time of coincubation of spermatozoa with the oocytes, differences in fertilization rates were seen among the various oviductal fluid treatments. At 14 and 16 h of coincubation, more oocytes were fertilized if spermatozoa were preincubated in isthmic oviductal fluid and oocytes were preincubated in ampullary oviductal fluid than if both gametes were incubated in whole or ampullary oviductal fluid. Because both sperm binding to the zona pellucida and fertilization rates were greater in treatments in which gametes were exposed to regional oviductal fluid than when they were incubated in whole oviductal fluid, it is concluded that oviductal fluid from different regions of the bovine oviduct may play different roles in facilitating fertilization.