Our experiments conducted under controlled laboratory conditions demonstrate diel uptake and release of zinc (Zn) in lab-cultured biofilm exposed to Zn concentrations that are present in some mining-impacted streams (1–2 mg Zn/l). Specifically, at constant pH, temperature, and aqueous Zn concentrations in the exposure water, biofilm accumulated Zn during the light periods of the photocycle and released Zn during the dark periods of the photocycle. The range of Zn uptake measured in biofilm during one light period in these laboratory experiments (0.6–8.3 mg Zn/g dw biofilm) encompassed the estimated Zn uptake (1.5–3.7 mg Zn/g dw biofilm) necessary to attribute aqueous diel Zn cycling in a mining-impacted stream in Montana (High Ore Creek) to uptake in biofilm. This is relevant to in situ studies of diel Zn cycling because we controlled three important parameters that naturally fluctuate daily in the field, thus demonstrating the potential for biofilm to remove large percentages of Zn from some mining-impacted streams. Researchers, modelers, regulators, and reclamation teams working in metals-contaminated streams should be aware of diel metal cycling, because the highest Zn concentrations (and therefore, perhaps the most toxic conditions) in the water column might occur at night, and the greatest exposure of grazers of phototrophs to dietborne Zn might occur during daylight hours.