Background: Feeding breastmilk to premature infants decreases morbidity but is often limited owing to an insufficient milk supply and delayed attainment of lactogenesis stage II. Early initiation of milk expression following delivery has been shown to increase milk production in mothers of very low-birth-weight (VLBW) infants. Although recommendations for milk expression in this population include initiation within 6 hours following delivery, little evidence exists to support these guidelines. This study compared milk volume and timing of lactogenesis stage II in mothers of VLBW infants who initiated milk expression within 6 hours following delivery versus those who initiated expression after 6 hours. Subjects and Methods: Forty mothers of VLBW infants were grouped according to when they initiated milk expression following delivery. Group I began milk expression within 6 hours, and Group II began expression after 6 hours. Milk volume was measured daily for the first 7 days and on Days 21 and 42. Timing of lactogenesis stage II was determined through mothers' perceptions of sudden breast fullness. Results: Group I produced more breastmilk during the initial expression session and on Days 6, 7, and 42. No difference in timing of lactogenesis stage II was observed. When mothers who began milk expression prior to 1 hour following delivery were removed from analysis, benefits of milk expression within 6 hours were no longer apparent. Conclusions: Initiation of milk expression within 6 hours following delivery may not improve lactation success in mothers of VLBW infants unless initiated within the first hour.