The bioaccumulation of methylmercury in fish is a complex process affected by many site-specific environmental factors. The US Environmental Protection Agency (USEPA) recently recommended changing the basis for expressing the ambient water quality criterion for mercury from an aqueous concentration to a measure of the methylmercury concentration in fish. This change would make the regulation of mercury in surface waters a site-specific exercise in which fish-based bioaccumulation factors (BAF; the ratio of mercury concentration in fish to the concentration of mercury in water) are used to calculate aqueous concentration limits for total mercury. These limits would then be used to allocate mercury loading among various point and nonpoint sources and guide regulatory actions. In order for this approach to succeed, it is critical that the site-specific BAFs and methylmercury:total mercury conversion factors be independent of aqueous total mercury concentration (HgT). We investigated this relationship by measuring aqueous methylmercury and HgTs and mercury in fish in ecologically similar warm-water streams in the southeastern United States. Bioaccumulation factors based on HgT in water were found to decrease with increasing HgT, primarily as a consequence of the reduction in the ratio of aqueous methylmercury to total mercury with increasing HgT. Methylmercury-based BAFs did not vary as a function of HgT. The implication of this relationship is that site-specific determination of aqueous HgT limits at contaminated sites may use BAFs that would be underestimates of the appropriate BAFs to describe mercury bioaccumulation in the system after mercury inputs have been reduced. In such cases, regulatory limits set using site-specific BAFs might not achieve their intended purpose of reducing mercury contamination in fish to acceptable concentrations.