Mercury (Hg) concentrations above levels that could pose health risks have been measured recently in predatory fish from many aquatic systems in the southeastern region of the United States. Based on hypotheses derived from published experimental data on the aqueous geochemistry of Hg, we investigated the effect of certain natural and human-imposed conditions on in situ levels of methylmercury (MeHg) in the Mobile-Alabama River System (MARS). Water samples were collected from different types of environments, hypothesized to have contrasting levels of MeHg in the aqueous phase, and were analyzed for total-Hg (THg) and MeHg concentrations, as well as some key geochemical parameters. The results showed the following. i) Overall, total Hg concentrations in waters of the MARS are quite uniformly distributed and vary from 0.2 to 6 ng L−1, suggesting that besides geological sources, atmospheric deposition is certainly the main source of Hg inputs in the studied system. ii) In locations with comparable THg levels, the Hg fraction present as MeHg was consistently higher in samples collected from the Coastal Plain portion of the MARS as compared to those from other geological provinces. iii) Our in situ observations confirmed conclusions derived from laboratory experiments, in that, MeHg abundance in aquatic systems correlates with sulfate (but only within a narrow range of concentrations); decreasing pH; and has no direct relationships with either nitrate or phosphate. iv) The investigation of Hg accumulation in biota at a single site showed that an aquatic system with low THg concentrations but a high MeHg:THg ratios, could have organisms with Hg content above safe levels. Therefore, potential health risks to fish eating populations can exist even when the aqueous phase does not show signs of significant Hg enrichment.