The Colorado River, Grand Canyon is a highly regulated 362-kilometer reach below Glen Canyon Dam. Dissolved organic carbon (DOC) input from Lake Powell is currently one of the largest fluxes of carbon to the downstream reach of the river, yet little is known about the dynamics of DOC in the system. The objective of this study was to quantify the fate of DOC in the Colorado River by assessing its sources using δ¹³C and quality of DOC. Potential DOC quality was measured using in-situ incubations of filtered stream water where the decrease of DOC is measured over time. Results indicate that DOC in the upper reach of the Colorado River below Glen Canyon (river kilometer 0), which is a highly productive reach of the river, is more bioavailable than DOC at further downstream sites. DOC concentration declined from 2.7 to 2.2 mgC/L from river kilometer 0 to 362. DOC-¹³C also decreased along the reach and was correlated with the potential bioavailability of DOC. It appears that little DOC is derived from tributaries at base flow and the decline of DOC concentration, DOC-¹³C signatures, and potential bioavailability along the 362 km reach are a result of in-stream processing along the reach.