Basic characteristics of European bison (Bison bonasus) semen were described and the efficacies of two extenders—Triladyl, containing egg yolk, and a synthetic extender, containing soybean lipids—were tested for semen cryopreservation. Seven ejaculates were collected by electroejaculation from a 10-yr-old, European bison bull. Each ejaculate was diluted at 37°C to a final concentration of 200 × 10⁶ sperm/ml with Triladyl or the synthetic extender. Extended semen samples were frozen according to a standard bull semen freezing protocol. After 2 wk of storage, one straw from each extender and ejaculate was thawed, and postthaw quality was evaluated by individual sperm motility and movement rate, numbers of sperm morphologic abnormalities and intact acrosomes, functional integrity of the sperm membranes determined by hypoosmotic swelling test (HOST), viability (live–dead, eosin–nigrosin stain), and a heterologous in vitro sperm penetration assay (SPA). A total of 600 in vitro–matured bovine oocytes were inseminated with 1 × 10⁶ spermatozoa of Holstein semen frozen–thawed in Triladyl (control) or of European bison semen frozen in Triladyl or the synthetic extender. Nuclear status of the oocytes was determined after 18 h of sperm–oocyte coincubation. Extender had no effect on any evaluated parameters of semen after dilution and cooling (4 hr at 5°C) or in postthaw individual motility, quality of movement, and sperm morphology. However, significantly (P < 0.05) higher numbers of spermatozoa with intact acrosomes, intact membranes (HOST), and viable sperm (P < 0.01) were in semen frozen in Triladyl than in the synthetic extender. Mean values for heterologous SPA for bull (control) and for bison semen frozen in the synthetic extender were very much alike—63.3 ± 10.6% and 63.1 ± 15.9%, respectively; bison semen frozen in Triladyl was lower, 43.0 ± 24.2% but not significantly different. Cumulative results from a variety of viability assays of diluted/cooled and frozen–thawed semen, including the heterologous SPA, suggest that European bison semen can be successfully frozen in both extenders tested in this study.