Variation in concentrations of acid‐volatile sulfide (AVS) in sediments from the upper Clark Fork River of Montana, USA, was associated with differences in bioaccumulation of Cu and Zn and growth of larvae of the midge, Chironomus tentans. Growth of midge larvae was significantly greater and bioaccumulation of Cu was significantly less in surface sections (0–3 cm depth) of sediment cores, which had greater concentrations of AVS and lesser ratios of simultaneously extracted metals to AVS (SEM:AVS ratios) than in subsurface sediments (6–9 cm). Concentrations of AVS were significantly less in sediments incubated with oxic overlying water for 9 weeks than in the same sediments incubated under anoxic conditions. Bioaccumulation of Cu differed significantly between incubation treatments, corresponding to differences in concentrations of AVS and SEM:AVS ratios, although midge growth did not. Bioaccumulation of Zn did not differ significantly between depth strata of sediment cores or between incubation treatments. When results from the two sets of bioassays were combined, bioaccumulation of Cu and Zn, but not growth, was significantly correlated with SEM:AVS ratios and other estimates of bioavailable metal fractions in sediments. Growth of midge larvae was significantly correlated with bioaccumulation of Zn, but not Cu, suggesting that Zn was the greater contributor to the toxicity of these sediments. Assessments of the toxicity of metal‐contaminated freshwater sediments should consider the effects of spatial and temporal variation in AVS concentrations on metal bioavailability.