Interaction of spermatozoa with oviductal epithelial cells (OEC) in the oviductal isthmus prolongs the life span of spermatozoa. The hypothesis that the interaction of equine spermatozoa with OEC affects their intracellular calcium concentration ([Ca2+]i) was tested in a sperm-OEC coculture model. Changes in [Ca2+]i in spermatozoa loaded with the fluorescent calcium indicator indo-1 acetoxymethylester (AM) were determined for spermatozoa attached to OEC or to Matrigel, as well as for free-swimming spermatozoa incubated without oviductal epithelium. [Ca2+]i was determined before incubation and at 0.5, 2, 4, and 6 h of incubation by ratio image analysis of fluorescent images captured at 405 nm and 490 nm. At each time point, [Ca2+]i was lower in motile spermatozoa attached to OEC than in free-swimming spermatozoa. [Ca2+]i in spermatozoa attached to Matrigel was lower than in free-swimming spermatozoa and was comparable to [Ca2+]i in spermatozoa attached to OEC only at 0.5 h incubation. Beyond 0.5 h of incubation, [Ca2+]i was higher in spermatozoa attached to Matrigel than in spermatozoa attached to OEC. These results indicate that spermatozoa with low [Ca2+]i might preferentially attach to OEC and to Matrigel, but that [Ca2+]i is maintained at this basal level only in spermatozoa attached to OEC. The reduced [Ca2+]i in spermatozoa associated with OEC may prevent premature capacitation and acrosomal exocytosis of spermatozoa stored in the isthmic sperm reservoir.