Oviduct epithelial cell (OEC) monolayers were prepared from the isthmic and ampullary parts of the oviducts of FSH-primed tammar wallabies. Co-culture experiments found that 50–60% of wallaby spermatozoa attached immediately to OEC monolayers, tracheal cell monolayer controls, and the surface of culture dishes with and without Matrigel coating. Spermatozoa were considered to be attached if they remained on the culture surface after rapidly pipetting the co-culture medium five times. The percentages of attached and unattached spermatozoa were calculated from the number of spermatozoa recovered in the agitated supernatant. After 2 h co-culture the percentage of attached spermatozoa rose to 60–80%. After 6 h co-culture the number of spermatozoa attached to OEC monolayers derived from the oviductal isthmus remained high and only a small percentage were recovered in the agitated supernatant (unattached spermatozoa 3.85 ± 0.76%, P = 0.67). However, after 6 h co-culture of spermatozoa with OEC monolayers derived from the ampulla and with the controls the percentage of attached spermatozoa declined significantly (unattached spermatozoa: ampullary monolayer 23.08 ± 4.80%, P < 0.01; tracheal monolayer 23.23 ± 5.18%, P < 0.01; Matrigel 27.23 ± 7.76%, P < 0.01; plastic surface 28.19 ± 5.30%, P < 0.01). After 6 h co-culture with ampullary and isthmic OEC monolayers, the percentage motility of both attached and unattached spermatozoa was maintained at 64.00 ± 1.90% and 56.66 ± 3.18% and 62.00 ± 3.11% and 52.00 ± 2.43%, respectively, and was then maintained at ≥ 35% after 24 h incubation. In the controls, that is, tracheal monolayer and Matrigel, the motility of attached spermatozoa declined rapidly to 48.66 ± 2.15% and 33.63 ± 8.66%, respectively, at 6 h, and all spermatozoa were immotile after 24 h incubation. However, the motility of unattached spermatozoa in the controls (tracheal monolayer and Matrigel) was maintained at 57.33 ± 3.00% and 34.54 ± 9.27%, respectively, until 6 h and then declined rapidly, and all spermatozoa were immotile after 24 h incubation. Co-culture of wallaby spermatozoa with OEC monolayers also induced acrosomal modifications that were followed by acrosomal loss. At 6 h incubation 38.92 ± 3.98% of spermatozoa on ampullary OEC monolayers and 36.50 ± 3.81% spermatozoa on isthmic OEC monolayers had shed their acrosome. Acrosomal loss during co-culture with both isthmic and ampullary OEC monolayers was significantly (P < 0.01) greater than that observed on tracheal epithelial monolayer (24.42 ± 1.90%, P < 0.01), Matrigel (20.70 ± 2.71%, P < 0.01) and plastic (15.54 ± 2.49%, P < 0.01). Co-culturing spermatozoa with OEC monolayers also induced a transformation from streamlined orientation of sperm head and tail to T-shaped (thumbtack) orientation in a small number (10–15%) of motile spermatozoa after 6 h incubation (data not shown). The significance of these results in relation to the role of the oviduct in sperm capacitation is discussed.