During fasting, the major glucose source switches to gluconeogenesis (GNG) which produces glucose from small metabolites such as lactate, glycerol and amino acids. It is believed that lactate is the major substrate contributing to GNG. However, lactate is largely generated from glucose and resynthesized to glucose, making lactate the largest direct contributor to glucose carbon but not necessarily a good source for new carbon entering GNG. Wang et al. fasted mice for 6, 12, and 18 hours to study the relative contribution of different GNG substrates. They demonstrate that lactate is the dominant direct contributor but a minor overall net carbon contributor to GNG. Instead, glycerol is the dominant net carbon source for GNG during short and prolonged fasting.