The purpose of this study was to evaluate plasmapheresis as a method for plasma extraction in comparison with centrifugation or gravity sedimentation. The study was designed as a cross over trial with six Freiberger horses undergoing plasma donation by plasmapheresis followed by whole-blood donation and subsequent plasma production 4 weeks later. Automated plasmapheresis and whole-blood donation were well tolerated in all horses. The plasmapheresis method achieved an almost complete removal of erythrocytes and leucocytes from plasma at all flow rates. After blood bag centrifugation, significantly more erythrocytes (P < 0.01) and leucocytes (P < 0.01) were present in the prepared plasma compared with plasmapheresis plasma. Plasma prepared by gravity sedimentation contained significantly more erythrocytes and leucocytes than plasma prepared with the other techniques (P < 0.01), and platelet aggregation was observed. The coagulation proteins and the total protein content of plasma prepared after plasmapheresis and blood bag centrifugation did not differ significantly from values measured prior to collection (P > 0.05). However, the activity of factor VIII was significantly lower 24 h after gravity sedimentation of blood than activity prior to blood collection (P < 0.01). In conclusion, automated plasmapheresis is the method of choice for the production of high quality equine plasma.