The role of biodegradation in the attenuation of natural organic matter (NOM) was investigated in long-term experiments that simulate artificial ground water recharge (AGR) for drinking water production. Lake water containing 5.8 mg L(-1) total organic carbon (TOC) was continuously fed into an 18.5-m-long sand column. During the 941 d of operation, on average 76 and 81% of TOC was removed within the first 0.6 m and the entire column length, respectively. Large molecular size fractions (approximately 1800-2200 Da) of NOM were removed more efficiently than smaller ones (approximately 250-1400 Da). The biodegradation of dissolved organic carbon (DOC) within the first 0.6 m, measured by the stable inorganic carbon isotope (delta13C) method, depended on temperature and hydraulic load: The extent of mineralization was 32% at 6 degrees C (Day 442) and 38% at 23 degrees C (Day 708) with a 0.3 m3 (m2d)(-1) hydraulic load and 52% at 5.5 degrees C (Day 883) with a 3.1 m3 (m2d) (-1) hydraulic load. The rest of the DOC removal was likely due to entrapment or sorption onto the sand particles. Decreases in DOC and the total cell counts in the water along the column were positively correlated (r = 0.99; P = 0.001). The accumulation of biomass was minor, with the highest concentration amounting to 7.2 mg g(-1) dw of sand. In summary, this study demonstrated that biodegradation has a key role in NOM removal in AGR and is dependent on temperature.