Microbial mats have been developed to sequester heavy metals from contaminated water. Mixed populations of photosynthetic and heterotrophic bacteria, dominated by SciUatoria spp., were developed for metal tolerance and integrated into a durable, self-sustaining community of microbes stimulated by and attached to ensiled grass. The mat was immobilized on glass wool and layered in flow-through baffled tanks. After allowing 8 weeks for the maturation of the mat, mixed solutions of Zn and Mn (15-16 mg L-i) were passed through a three-tank experimental series. Effluent from each tank was first sampled and then applied to the next tank. This procedure was repeated in triplicate and with six applications of new metal solution per three-tank series. By the third tank, the target metal concentration < 1 mg L-1 was always achieved. Mean percentages of the initial influent concentration removed by tanks 1, 2 and 3, respectively, were 72, 93 and 98 for Zn and 78, 97 and 99 for Mn. Mean metal concentrations in the effluents (average of 6 applications) were, for tank 1: Zn (mg L-l) 5.0, Mn (mg L -1) 4.2; for tank 2: Zn 1.6, Mn 0.75; for tank 3: Zn 0.53, Mn 0.19. Mean effluent concentrations from each of the three sequential treatments (average of 6 applications per tank) were for Zn (mg L-1) 5.0, 1.6 and 0.53; for Mn (rag L -l) were 4.2, 0.75 and 0.19. Thus target concentrations were reached in experimental tank 2 for Mn and tank 3 for Zn. Metal removal in the control tank series, containing glass wool only, was 37% for Zn and 5% for Mn (average of 6 applications). Oxygen and redox potential analyses of the mat/glass wool matrix revealed a heterogenous structure of anoxic and oxic zones. Zeta potential analysis of the mat samples identified a mat surface charge ranging from -12.3 to -69.2 inV. Various metal removal mechanisms possibly involved with metal sequestering include surface binding to the mat or to mat exudates trapped within the glass wool, precipitation of the metals with anions present in the oxic/anoxic zones, mat mediation of the water conditions in favor of metal-oxide precipitation and active transport of the metals into the cell.