Red clover (Trifolium pratense L.) silage usually contains lower contents of non-protein nitrogen (NPN) compared with other forage legumes. This is often attributed to the polyphenol oxidase (PPO) activity in red clover, although in most field studies the PPO activity was not measured. Therefore, a laboratory ensiling experiment with three red clover cultivars and one white clover cultivar as control grown in two management systems (with and without mechanical stress) over 2 consecutive years was conducted. Fresh, wilted and ensiled clover herbage was sampled at four cutting dates per year to determine the crude protein (CP) fractions according to the Cornell Net Carbohydrate and Protein System. The specific PPO activity was measured photometrically in fresh clover leaves. The content of CP fraction A (NPN) increased from fresh over wilted to ensiled clover herbage at the expense of the content of CP fraction B (true protein), irrespective of species, cultivar and year. The most important source of variation for all CP fractions and the calculated rumen-undegradable protein contents was generally the herbage condition, except for CP fraction C (unavailable protein). White clover silage consisted of higher contents of CP fraction A and lower contents of CP fraction B3 in CP compared with red clover silage. As a result, the calculated rumen-undegradable protein content of white clover silage was lower than that of all red clover cultivars. In conclusion, the extent of proteolysis during ensiling among the silages made from the herbage of different red clover cultivars was primarily influenced by the stage of maturity at harvesting and the degree of wilting at ensiling. The variation in specific PPO activity could not be related to the extent of proteolysis.