Two small drainage lakes of similar surface area (0.4–0.6 km2) and depth, and situated in close proximity to one another (about 9 km apart) in the Muskoka-Haliburton district of rural Ontario, Canada were chosen to investigate the effects of differing pH on Hg accumulation in fish and other biota. Blue Chalk Lake is circumneutral (pH = 6.3–6.9) with an average dissolved organic carbon (DOC) concentration of 2.69 mg/L; Plastic Lake is acidic (pH = 5.2–5.6) with a similar DOC level (2.72 mg/L). Neither lake is an impoundment, and neither receives inputs from point-source urban or industrial waste emissions. Surficial (top 4 cm) sediments and various aquatic biota, including crayfish (Orconectes virilis)], and 5 species of fish [brown bullhead (Ictalurus nebulosus), creek chub (Semotilus atromaculatus), pumpkinseed sunfish (Lepomis gibbosus), yellow perch (Perca flavenscens) and rock bass (Ambloplites rupestris)] were sampled for Hg analysis. Only creek chub and pumpkinseed occurred in both lakes. Significant size (length) vs. Hg-concentration relationships were observed for brown bullhead, pumpkinseed, yellow perch and rock bass, but not for crayfish nor for creek chub in either lake. Rock bass had the highest mean Hg concentration (1.02 ± 0.98 μg/g dry wt.). Pumpkinseed from Plastic Lake had the highest overall rate of accumulation with increasing size. Several individual pumpkinseed and rock bass sampled from the acidic Plastic Lake had Hg concentrations equal to or greater than levels reported to be hazardous to breeding piscivorous birds, such as common loons (Gavia immer), whereas no fish from Blue Chalk approached this threshold (0.3 μg/g wet wt). This study illustrates the importance of within-lake parameters, particularly pH, and species trophic level, in determining Hg accumulation in fish in lakes of similar size that receive similar atmospheric Hg inputs.