The percentage dose of lactulose and mannitol excreted in urine after oral ingestion is used as a noninvasive method of assessing small intestinal permeability. The collection of incomplete or inaccurately timed urine samples can lead to errors in estimation of sugar probe molecules. We describe an HPLC method for the simultaneous determination of lactulose and mannitol in serum after oral ingestion of test sugars. We applied the test to healthy volunteers and to subjects undergoing jejunal biopsy for suspected gluten-sensitive enteropathy. The ratio of concentrations of lactulose and mannitol in serum discriminated well between subjects with a normal biopsy and those with villous atrophy, discrimination being best at 90 min postdose. The results agree well with lactulose:mannitol ratios determined in urine (r= 0.88), and the two methods can be used interchangeably. The determination of mannitol and lactulose in serum provides an acceptable alternative to urine collection and may be particularly useful in young children. It also reduces the time spent on the investigation from 5 h to 90 min.