The vacuum in the mouthpiece chamber (MPC) was measured in a factorial trial with two liners (DeLaval 964008-01 and DeLaval 999007-03), two pulsator ratios (60 and 70%) and morning and evening milking over eight milkings of ten cows. Teat length and diameter were measured before and after milking, and maximum milk flow rate and depth of penetration of the teat into the teatcup were also recorded. The MPC vacuum at peak flow rate was classified as stable, at various levels, in 79% of the recordings. In the rest of the milkings the vacuum declined gradually, and this decline was associated with gradually deeper penetration of the teat into the teatcup. The DeLaval 964008-01 liner with the narrower bore and larger mouthpiece opening gave lower MPC vacuum at peak flow rate than did the wider bore DeLaval 999007-03 with the smaller mouthpiece opening. Morning milking gave higher MPC vacuum than evening milking. Increased maximum milk flow rate and increased length and diameter of the teat gave decreased MPC vacuum. The teats were longer before milking in the morning than in the evening. However, during peak flow rate, the teats had penetrated deeper into the teatcup in the evening than in the morning. A marked change in MPC vacuum in the transition to the period of flow rate can be used to identify the start of over-milking of an individual teat. A general model explaining the MPC vacuum as the outcome of the balance between air leakage past the mouthpiece lip and the leakage past the teat in the liner barrel is suggested.