Anaerobic transformations of wastewater organic matter in the bulk water phase of collection system networks were investigated in laboratory-scale experiments. The wastewater was collected from three locations, which provided samples with different characteristics, ranging from young to mature. Hydrolysis, fermentation, and sulfate reduction were identified as the most important anaerobic processes. Significant quantities of readily biodegradable substrate were produced by hydrolysis of complex organic substrates. The readily biodegradable substrate was further fermented into volatile fatty acids (VFA). The rate of fermentation was found to be limited by the hydrolysis process. The readily biodegradable substrate generated was almost entirely composed of VFA, primarily acetic and propionic acids. A production of sulfide was observed in all experiments, demonstrating that part of the readily biodegradable substrate was consumed by sulfate respiration. The sulfide production was most pronounced in mature wastewater that had previously undergone extended anaerobic transport.