Carbohydrate energy absorption and breath hydrogen concentration were measured in 12 premature infants 28-32 wk gestational age and 2-4 wk postnatal age. Each of two groups of six infants were randomly assigned to receive one of two formulas that differed only in carbohydrate source: 100% lactose (LAC) or 50% lactose: 50% glucose polymer (LAC + GP). In 11 infants the peak breath hydrogen concentration suggested extensive colonic fermentation (range 44-239 ppm/5% CO2 or 44-239 microL/L per 50 mL/L CO2). An approximate 100% increase in lactose intake in the LAC group was associated with a similar increase in breath hydrogen concentration at 30, 60, and 120 min. None of the infants exhibited diarrhea or vomiting or developed delayed gastric emptying. Carbohydrate energy absorption (mean +/- SD) was, respectively, 86 +/- 5% and 91 +/- 3% in the LAC and the LAC + GP groups (p greater than 0.05). Thus, colonic bacterial fermentation may be critical to energy balance and to the prevention of osmotic diarrhea in premature infants fed lactose.