The proportions of milk stored in the gland cistern (cisternal) and within secretory alveolar tissue (alveolar) were determined for a group of ten cows in mid lactation, yielding ∼ 20 kg milk/d. As a proportion of total milk, cisternal milk, measured at 8 h after milking, ranged from 0·09 to 0·53. Milking frequency was then reduced from twice daily to once daily on two occasions, first for 1 week and then, 2 weeks later, for 2 weeks. Milk yield was reduced during once daily milking in all cows, by an average of 22·8% during the first week (P < 0·001). The amount of decrease varied among cows in a way that was unrelated to pretreatment yield, but the immediate decrease was significantly and inversely correlated with cisternal milk proportion (r = 0·81, P < 0·01). When milking reverted to twice daily, yield recovered to a value not significantly different from pretreatment, and the degree of recovery was inversely related to cisternal milk proportion (r = 0·74, P < 0·01). With the exception of one cow, yield decreases correlated well between the two periods of once daily milking. When treatment weeks were disregarded, lactation persistency (the rate of decline in yield with time) was unaffected by once daily milking. The results show that the ability of individual cows to tolerate once daily milking is related to their cisternal storage characteristics; yield is reduced less in cows that store a greater proportion of their total milk production within the cistern. Once daily milking for up to 2 weeks had no lasting effect on milk yield or lactation persistency, suggesting that there had been no detrimental effect on udder development.