Concentrations of microbial ATP in 15 New Zealand lakes were determined using 2 extraction techniques. ATP was found in both the paniculate (>0.45 μm) and dissolved (<0.45 μm) fractions of the water samples. Higher concentrations of paniculate ATP were obtained when ATP was extracted directly from freshwater samples rather than following pre‐concentration on to membrane filters. Recovery of particulate ATP declined with increasing volumes of sample filtered. The magnitude of the filtration effect varied unpredictably between lakes and within lakes with time. Corrections for the presence of dissolved ATP were necessary before estimates of particulate ATP could be made by the direct extraction technique. Concentrations of dissolved ATP up to 1.2 μg L‐1were observed. The direct extraction technique is recommended for more accurate determination of particulate ATP in New Zealand freshwaters.