Background The bileaflet valve is currently the mechanical replacement valve of choice. Though durable, it does not closely mimic native valve hemodynamics and remains potentially thrombogenic. Methods Prototype trileaflet valves (T1 and T2) were implanted in the mitral position in calves. Group I calves received either a T1 valve (n = 12) or a control bileaflet valve (n = 5); Group II, either a T2 valve (n = 7) or a control bileaflet valve (n = 5). Valve function, perivalvular leakage, and transvalvular pressure gradients were evaluated. Also, long-term prototype leaflet wear was evaluated in vivo in one Group I calf (502 days) and two Group II calves (385 and 366 days). Calves were euthanized and necropsied at study termination, and major organs weighed and examined. Results Valve function was excellent and hematologic parameters remained normal in all calves that survived to study termination. Mean peak transvalvular pressure gradients were 10 ± 7 mm Hg for T1 valves, 6 ± 3 mm Hg for T2 valves, and 12 ± 4 mm Hg for bileaflet control valves. Clinically insignificant valvular regurgitation was observed in both prototypes. Explanted valves showed no thrombus-impaired leaflet motion, except in two T1-fitted calves and one T2-fitted calf. Major organs showed no evidence of clinically significant thromboembolic events. There were no other significant differences between the results of experimental and control groups. Conclusions Prototype trileaflet valves performed safely and effectively in the mitral position in calves, even without long-term anticoagulation. This warrants their evaluation as an equivalent alternative to bileaflet valves.