Interaction of equine spermatozoa with oviductal epithelial cells (OEC) prolongs sperm viability and maintains low intracellular calcium concentration ([Ca²⁺]_i) in spermatozoa. Experiments were designed to investigate 1) whether release of spermatozoa from OEC in vitro is associated with elevated [Ca²⁺]_i and 2) whether soluble products from OEC or direct membrane contact between spermatozoa and OEC mediates the effects of OEC on sperm [Ca²⁺]_i. In the first experiment, changes in [Ca²⁺]_i in spermatozoa loaded with indo-1 acetoxymethylester were determined in motile spermatozoa released from OEC monolayers after 4 h of culture compared to [Ca²⁺]_i in spermatozoa still attached to OEC. In addition, [Ca²⁺]_i was determined in spermatozoa incubated with OEC-conditioned medium for 6 h compared to that in spermatozoa incubated in control medium. [Ca²⁺]_i was higher in motile spermatozoa released from OEC than in spermatozoa still attached to OEC after 4 h of incubation. Incubation in OEC-conditioned medium resulted in lower sperm [Ca²⁺]_i only at 4 h of incubation, but not at 0.5, 2, or 6 h of incubation. In the second experiment, a suspension of apical plasma membrane vesicles (AMV) isolated from isthmic oviductal epithelium was used to study the specific effect of sperm contact with OEC membranes on sperm viability, capacitation, and [Ca²⁺]_i. Direct membrane contact between spermatozoa and AMV prolonged sperm viability, delayed capacitation, and maintained low [Ca²⁺]_i in spermatozoa. These results indicated that membrane contact between equine spermatozoa and OEC is required to maintain low [Ca²⁺]_i, delay capacitation, and prolong viability of spermatozoa in vitro. Modulation of capacitation rate for spermatozoa stored in the isthmic sperm reservoir might ensure the availability of a competent sperm population at the time of fertilization.