CLSM and fluorescent probes were applied to assess the structure, composition, metabolic activity and gradients within naturally occurring β-proteobacteria microcolonies. Extracellular polymeric substances (EPS) as defined by lectin-binding analyses had three regions: (i) cell associated, (ii) intercellular and (iii) an outer layer covering the entire colony. We assessed structural, microenvironmental and metabolic implications of this complex EPS structure. Permeability studies indicated that the outer two layers were permeable to 20 nm beads, intercellular EPS to <40 nm beads and the outer layer was permeable to <100 nm beads. Phosphatase activity occurred at the cell surface and associated polymer. Glucose oxidase activity was only detected inside the cells and the cell-associated polymer. Rhodamine 123 suggested that activity was highest near the cell surface. The potential sensitive dye JC-1 concentrated within the outer EPS layer and the gradient was responsive to inhibition by KCN, dispersion using KCl and enhanced by addition of nutrients (nutrient broth). pH gradients occurred from the cell interior (pH 7) to the microcolony interior (pH 4+) with a gradient of increasing pH (pH 7+) to the colony exterior. The EPS provides a physical and chemical structuring mechanism forming microdomains that segregate extracellular activities at the microscale, possibly resulting in a microcolony with unitary structure and function.