A total of 20 water samples collected from the cooling towers at 20 different sites were analyzed under various conditions for the presence of Legionella pneumophila serogroup 1. A comparative assessment was performed to evaluate methods of sample collection (spray drops, beneath water at 20- to 40-cm depth, and water outlet), concentration (filtration and centrifugation), acid buffer treatment (no treatment, treatment for 3, 5, and 15 min), and CO2 incubation or candle jar incubation. The reduction in viable colonies and false negative rate were compared for the different factors. No quantitative differences in isolation of L. pneumophila serogroup 1 was found among samples collected from water at a depth of 20 to 40 cm, from water outlet, and from spray drops. Treatment in an acid buffer for 15 min significantly reduced the recovery rate, with a reduction in bacterial counts of about 40%, compared with a 3-min (12%) or a 5-min (25%) treatment. Acid buffer treatment for 3 or 5 min reduced the overgrowth of commensal flora. This treatment improved the selectivity but not the sensitivity for L. pneumophila serogroup 1. Colonies on plates incubated at 37 degrees C in a candle jar with a humidified atmosphere grew better than those incubated at 35 degrees C with 5% CO2. These results demonstrate that methods of sample collection, concentration, and incubation, but not collection site, can affect the isolation rate for L. pneumophila serogroup 1.