We report a new method to measure the fraction of glucose derived from gluconeogenesis using gas chromatography mass spectrometry and positive chemical ionization. Following ingestion of deuterium oxide, glucose derived from gluconeogenesis is labeled with deuterium. Our calculations of gluconeogenesis are based on measurement of the average enrichment of deuterium on the 1, 3, 4, 5 and 6 carbons of glucose and the deuterium enrichment in body water. In a sample from an adult volunteer following ingestion of deuterium oxide, fractional gluconeogenesis using the "average deuterium enrichment method" was 48.3±0.5% (Mean ± SD) and that of the C-5 HMT method by Landau et al. was 46.9±5.4%. The coefficient of variation (CV) of 10 replicate analyses using the new method was 1.0% compared to 11.5% for the C-5 HMT method. In samples derived from an infant receiving total parenteral nutrition, fractional gluconeogenesis was 13.3±0.3% using the new method and 13.7±0.8 % using the C-5 HMT method. Fractional gluconeogenesis measured in six adult volunteers after 66 h of continuous fasting was 83.7±2.3% using the new method and 84.2±5.0% using the C-5 HMT method. In conclusion, the "average deuterium enrichment method" is simple, highly reproducible and cost effective. Further, it requires only small blood sample volumes. Using an additional tracer, glucose rate of appearance can also be measured during the same analysis. Thus, the new method makes measurements of gluconeogenesis available and affordable to large numbers of investigators under conditions of low and high fractional gluconeogenesis (~10 to ~90) in all subject populations.