The amount of crude protein in white clover solubilised during ingestion by cows was determined by collecting swallowed boluses in rumen fistulated cows. Six experiments were then conducted to determine the effects of sample preparation on in situ estimates of rumen degradability of crude protein in white clover (Trifolium repens L.) herbages. Different methods of preparation were compared with chopped fresh herbage, a recommended method when this type of feed is incubatedin situ. Samples of white clover were either: (i) chopped fresh to 10 mm lengths (FRS), (ii) frozen, freeze-dried and ground (FRZ/D), (iii) oven-dried at 45°C for 48 h and ground (OD45C), (iv) oven-dried at 60°C for 48 h and ground (OD60C), or (v) minced fresh in a food processor (MNC). The immediate solubilities of crude protein from MNC preparations most closely approximated measures of the proportion (0.28–0.37) of crude protein solubilised during ingestion by cows, while immediate solubilities of crude protein from FRS preparations were low (0.09–0.19). The effective rumen degradability of protein for FRS was lower (P<0.05) than for MNC, FRZ/D or the oven-dried preparations. The effects of the method of preparation of fresh clover on estimates of rumen degradable protein and undegraded dietary protein were compared assuming cows were producing <15 L milk/day. In the 2 experiments comparing FRS and MNC preparations, the average estimates of rumen degradable protein and undegraded dietary protein were 158 and 83 g/kg DM, respectively, for FRS, compared with 189 and 52 g/kg DM for MNC. Over the 4 experiments comparing fresh and dried preparations, estimates of rumen degradable protein and undegraded dietary protein for FRS were 140 and 101 g/kg DM, respectively, and differed from those for FRZ/D of 156 and 70 g/kg DM. The implications of such differences have been discussed in relation to use of the in situ technique. It was concluded that no method of sample preparation will be ideal and it is likely the preferred method of sample preparation for in situ studies will vary for fresh herbages compared with conserved forages and concentrates.