The dynamics of dissolved organic carbon (DOC) have been intensively studied in mature ecosystems, but little is known about DOC dynamics and the significance of DOC as a substrate for microbial activity in early-successional catchments. We determined the concentration, chemical composition, source, radiocarbon age, and bioavailability of DOC along the hydrological flow path from soil solution to a downstream pond in a recently constructed catchment (Chicken Creek Catchment, Germany). Soil solution, upwelling ground water, stream water, subsurface water in an alluvial fan, and pond water all had high DOC concentrations (averages: 6.0–11.6 mg DOC L–1), despite small carbon stocks in both vegetation and soil of the catchment. Solid-state CPMAS 13C NMR of DOC in upwelling ground water revealed a higher proportion of aromatic compounds (32%) and a lower proportion of carbohydrates (33%) than in pond water (18% and 45%, respectively). The average 14C age of DOC in upwelling ground water was 2600 to 2900 yr, while organic matter of the Quaternary substrate of the catchment had a 14C age of 3000 to 16 000 yr. Both the 14C age data and 13C NMR spectra suggest that DOC partly derived from organic matter of the Quaternary substrate (about 40 to 90% of the C in the DOC), indicating that both recent and old C of the DOC can support microbial activity during early ecosystem succession. However, in a 70 day incubation experiment, only about 11% of the total DOC was found to be bioavailable. This proportion was irrespective of the water type. Origin of the microbial communities within the catchment (enriched from soil, stream sediment or pond water) also had only a marginal effect on overall DOC utilization.