The ratio of magnesium to calcium (Mg/Ca) in carbonate minerals in an abiotic setting is conventionally assumed to be predominantly controlled by (Mg/Ca)solution and a temperature dependant partition coefficient. This temperature dependence suggests that both marine (e.g. foraminiferal calcite and corals) and freshwater (e.g. speleothems and surface freshwater deposits, "tufas") carbonate deposits may be important archives of palaeotemperature data. However, there is considerable uncertainty in all these settings. In surface freshwater deposits this uncertainty is focussed on the influence of microbial biofilms. Biogenic or "vital" effects may arise from microbial metabolic activity and/or the presence of extracellular polymeric substances (EPS). This study addresses this key question for the first time, via a series of unique through-flow microcosm and agitated flask experiments where freshwater calcite was precipitated under controlled conditions. These experiments reveal there is no strong relationship between (Mg/Ca)calcite and temperature, so the assumption of thermodynamic fractionation is not viable. However, there is a pronounced influence on (Mg/Ca)calcite from precipitation rate, so that rapidly forming precipitates develop with very low magnesium content indicating kinetic control on fractionation. Calcite precipitation rate in these experiments (where the solution is only moderately supersaturated) is controlled by biofilm growth rate, but occurs even when light is excluded indicating that photosynthetic influences are not critical. Our results thus suggest the apparent kinetic fractionation arises from the electrochemical activity of EPS molecules, and are therefore likely to occur wherever these molecules occur, including stromatolites, soil and lake carbonates and (via colloidal EPS) speleothems.