Experiments were undertaken to validate a method (using adrenaline injection) for determination of the size of cisternal and alveolar compartments in the udder, to use this method to determine the pattern of milk accumulation in the udder over time and to determine the relationship between the size of the alveolar and cisternal compartments and tolerance of once daily milking. Cows received intrajugular injections of adrenaline (3 mg) immediately before milking, to block milk ejection and allow harvesting of the cisternal milk fraction. This was followed by removal of the alveolar fraction 30 min later after intrajugular oxytocin (5 i.u.) injection. Results obtained were similar to those obtained by catheter drainage. The alveolar compartment was 90% full at 16 h post milking while the cisternal compartment filled more slowly and was only 70% full at 24 h post milking. At full capacity (measured at 40 h), the volumes of milk contained in the cisternal and alveolar compartments were similar. In a further experiment involving identical twin cows, it was shown that the greater the degree of filling of the cisternal compartment at 24 h, the lower was the production loss on once daily milking. This suggests that the freedom of the alveoli to drain was an important factor in the production loss on once daily milking. Although there were significant correlations within twin sets for milk yield and the size of udder compartments, the relationship within twin sets for yield loss on once daily milking was not statistically significant.