The effects of replacing whole cottonseed by heated whole cottonseed at two levels of crude protein on amino acid (AA) utilization by the bovine mammary gland were investigated. Four Israeli Holstein cows were used in a 4 x 4 Latin square design with 2 x 2 factorial arrangements. Diets were formulated to contain two levels of crude protein (CP), normal (160 g CP/kg dry matter) and low (140 g CP/kg dry matter), and two levels of rumen undegradable protein (UDP), high (380 g UDP/kg CP) and low (350 g UDP/kg CP). Whole cottonseed was quantitatively substituted by heated whole cottonseed to formulate the high UDP diets. Intakes of dry matter and organic matter were similar for all treatments and averaged 15.9 and 14.4 kg/d respectively. Yields of milk and its constituents were similar for all treatments. Milk yield averaged 23.1 kg/d and this contained (per kg) 32.3 g CP, 25.4 g total casein and 47.5 g lactose. The milk fat content was lower in the cows given the high UDP diets, averaging 34.6 g/kg compared with 38.9 g/kg in the cows fed on the diets with low UDP. Plasma AA concentrations were similar for all treatments apart from Leu and Ile, which were higher in the cows given the high CP diets: 152 and 103 μM compared with 183.8 and 131.5 μM for the low CP diets. Net extraction and balance of essential AA across the half udder suggested that essential AA were supplied in amounts sufficient to meet milk protein requirements for all treatments. The surplus AA supplied as a result of feeding the high dietary CP were probably catabolized via an enhanced oxidative pathway, possibly in the liver, presumably as a passive response to their disposal.