One hundred and five healthy subjects under investigation for lactose malabsorption were enrolled in this prospective study. After an overnight fast, breath was collected immediately before and every 15 minutes for three hours after an oral administration of 50 g lactose dissolved in 200 mL of water (lactose overload). Blood samples were also collected before and 20, 40 and 60 minutes after the lactose overload for blood glucose determination. Expired H2 was measured by gas chromatography using a Quintron Microlyser and the maximum increase in H2 (delta-H2) was calculated. The area under the curve (ASC-H2) was also calculated using the trapezoid model. Lactose malabsorption was defined as a maximum increase in blood glucose (delta-G) under 18 mg%. The sensibility (S), specificity (E) and accuracy (A) of the H2 determination was analyzed using (a) linear regression [log (ASC-H2) = 4.034-0.029. delta-G], (b) receiver operating curve (ROC) and by (c) Q-Q plot of the cubit root transformation of the ASC-H2 values. Our results demonstrate that ASC-H2, but not delta-H2, correlates with delta-G, suggesting that the measurement of maximum increase in H2 expired following a lactose overload is not reliable for the diagnosis of lactose malabsorption. The cutoff value for the ASC-H2 using linear regression was 3260 (S = 89.3%, E = 80% and A = 86.7%), while using ROC it was 3353 (S = 88.0%, E = 80.0% and A = 85.7%) and while using Q-Q plot method it was 2936 (S = 92.0%, E = 80.0% and A = 88.6%). When the cutoff value was set to 3000, the sensibility, specificity and accuracy were 90.7%, 80.0% and 88.6%, respectively. We conclude that the calculation of the ASC-H2, but not delta-H2, following a lactose overload is reliable in detecting lactose malabsorption and can be used in the clinical practice.3618-26