Thirty-four mature Holstein cows were blocked into two groups on their 305-day, twice daily milked, mature equivalent milk production of the previous lactation; groups were assigned at random to a twice or thrice daily milking frequency. In addition, six pairs of paternal half-sisters and one pair of nonsibling heifers also were assigned to twice daily or thrice daily milking frequency. Half-sisters were assigned to milking frequency on alternate calvings, and the nonsibling pair was assigned at random. Milk production peaked at 6 wk lactation in mature cows in both treatments, at 7 wk for cows in first lactation milked twice daily, and at 9 wk for cows in first lactation milked thrice daily. Cows milked thrice daily reached higher peak milk production and were more persistent in milk production. Multiparous and first-lactation cows milked thrice daily produced 18.5 and 25.2% more milk than their counter-parts milked twice daily, and after 15 wk heifers milked thrice daily were producing more milk per day than mature cows milked twice daily. The combined increased average production for cows and heifers milked thrice daily, 1299kg, required only 92kg more dry matter intake and resulted in an apparent preferential utilization of feed nutrients for milk production or higher rate of tissue catabolism in cows milked thrice daily. Milk composition was not influenced by milking frequency; total milk fat was 36kg more for cows milked thrice daily. Reproductive performance measured as days to first estrus, days open, or services per conception also was not influenced by milking frequency.