Hummingbirds obtain most of their dietary calories from floral nectar ingested during hovering flight. Despite the importance of dietary sugar to hummingbird metabolism, the turnover of newly ingested carbon in the pool of actively metabolized substrates has not been adequately characterized in hovering hummingbirds. By combining respirometry with stable carbon isotope analysis of respired breath, we show that in rufous (Selasphorus rufus) and Anna's (Calypte anna) hummingbirds at high foraging frequencies, utilization of newly ingested sugars increased over a period of 30-45 min until it accounted for virtually 100% of the fuel oxidized. This newly ingested sugar disappears from the actively metabolized pool of substrates over a similar time course. These results demonstrate that turnover of carbon in the pool of actively metabolized substrates is rapid; carbon from ingested sucrose is available for oxidation for 30-45 min before being cleared. By monitoring expired CO2 for the appearance and disappearance of the signature characteristic of newly ingested sugar and then calculating energy budgets using video recordings of hummingbird activity, we estimated the proportion of recently ingested sugar used to fuel ongoing metabolism as well as the proportion devoted to energy storage. Consistent differences between species in the percentage of ingested cane sugar oxidized during the 2 h experimental periods suggest that individuals of each species adopted energy intake patterns appropriate to their needs. This approach provides a means by which to examine the partitioning of dietary carbon intake between energy expenditure and storage using non-invasive, field-compatible techniques.