Twelve multiparous British Friesian cows in early (40±23 d in milk; n=6) or late (216±17 d in milk; n=6) lactation were used to study the effects of milking frequency on yield, udder volume and milk storage within the udder. After a 2 week control period of twice daily milking, diagonally opposed udder halves within a cow were milked once or twice daily for 3 weeks. Milk yield was 28–38% lower from the halves that were milked once daily than from halves that were milked twice daily. The loss of milk yield, expressed as a decrease in the relative milk yield quotient (an index that accounts for pretreatment differences), was greater for cows in early than in late lactation (0·59 v. 0·68). Empty udder-half volume was not decreased by once daily milking, suggesting that no cell loss occurred. Instead, once daily milking reduced the secretion efficiency (units of milk per unit of empty udder-half volume) by 46 and 27% respectively in early and late lactation; thus, at least part of the loss was due to reduced metabolic activity of the mammary epithelium. There were positive correlations between the relative milk yield quotient and the proportion (r=0·804) or volume (r=0·644) of cisternal milk in the glands that were milked once daily. These results confirm that, during extended milking intervals, milk loss was smallest for cows that stored a larger proportion of milk in the gland cistern.