Ultrasonic nebulization of lactate dehydrogenase (LDH) was investigated using a DeVilbiss "Aerosonic" nebulizer. The enzyme (8ml, 0.025 mg/ml Na2HPO4, pH 7.0) was completely inactivated after 20 minutes of operation. However, the inactivation profile observed during ultrasonic nebulization was different from that previously observed using air-jet nebulization. At least two mechanisms are involved, one associated with heating and the other with aerosol production. By preventing heating of the nebulizer fluid during operation, the denaturation profile was dramatically altered. By additionally including 0.01% w/v Tween 80 or 1% w/v PEG 8000, almost all activity was retained. Similar results were obtained by preventing aerosol production and heating. However, 100% of activity was lost when heating was allowed to occur without aerosol formation. The results demonstrate that cooling in conjunction with a surfactant is one approach that could be used to stabilize proteins to ultrasonic nebulization. However, cooling also significantly reduced solute output from the nebulizer. When operated at 10 degrees C output was negligible. At 50 degrees C the output was 5x greater than that found at room temperature. The median droplet size (micron(s)) was not significantly influenced by the operating temperature of the nebulizer fluid (3.6 +/- 0.4, 21 degrees C; 3.9 +/- 0.2, 50 degrees C, p = NS (n = 6)) although the size distribution was noted to increase at the higher temperature.