High Hg concentrations in freshwater fish are a concern for human health, yet we lack a clear understanding of the mechanisms that produce high Hg concentrations in fish. Controlled studies in natural surface waters that quantify the uptake and retention of Hg in fish tissues following exposures from the aqueous phase and from invertebrate prey diets are rare. Using 203Hg, we contrasted the accumulation of inorganic Hg (HgI) and methylmercury (MeHg) from the dissolved phase and from invertebrate food in mosquitofish (Gambusia affinis) feeding on Daphnia pulex (representing a pelagic food chain) and in redear sunfish (Lepomis microlophus) feeding on amphipods (Hyallela sp., representing a benthic/macrophyte-based chain). Experiments were conducted with environmentally realistic Hg concentrations in two freshwaters from the San Francisco Bay Delta (CA, USA) with significantly different dissolved organic carbon (DOC) concentrations. Mercury uptake rates following aqueous exposures were consistently higher for fish in the water with lower DOC, whereas efflux rates were similar for both water types. Approximately 50% of the ingested HgI associated with invertebrate prey was lost from mosquitofish, and 90% or more from sunfish, within 48 h. Assimilation efficiencies for ingested MeHg for both fish were 86 to 94%, substantially higher than those for HgI regardless of water type. Biokinetic modeling using the parameters determined in these experiments accurately predicted Hg burdens for fish in the San Francisco Bay Delta system. Despite considerable accumulation of HgI from both aqueous and dietary exposure routes, the high assimilation efficiencies and slow loss of MeHg from dietary sources are the principal determinants of predicted Hg burdens in both fish species.