The stability of recombinant consensus alpha-interferon (rConIFN) to air-jet and ultrasonic nebulization was evaluated. Volumes of 10 mL of 0.5 mg/mL rConIFN in phosphate-buffered saline (PBS) at pH 6.3 were nebulized with a Collison three-jet nebulizer at 40 psig (10 L/min) for up to 25 min. The effects of pH (3.0, 6.3, and 9.0), additive (0.1% w/v Tween 80, 0.1% w/v Tween 20, and 1% w/v PEG 8000), and ionic strength (0, 0.25, and 1.0) were examined. The effects of ultrasonic nebulization were studied using three devices (DeVilbiss "Aerosonic"; Mountain Medical "Microstat", and Medix "Easimist"). Stability of rConIFN was assessed by size exclusion chromatography and native and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE). Structural effects were examined by circular dichroism spectroscopy and bioactivity was assessed by an in vitro mitogenic inhibition bioassay. rConIFN is destabilized by air-jet nebulization. Insoluble noncovalent aggregates are produced rapidly, and only approximately 25% of the initial monomeric protein remains after 25 min of nebulization. This correlates with a decrease in in vitro bioactivity. Aggregation during nebulization is influenced by pH (9.0 < 6.3 < 3.0) but even at the highest pH, > 25% aggregation is observed. Ionic strength does not appear to influence aggregation. rConIFN is also seen to adhere to glass after nebulization. Samples from a rinse of the emptied reservoir with 0.1% w/v SDS, after thorough rinsing with water (three times), show a strong rConIFN band on SDS-PAGE gels. The use of PEG 8000 and Tween mitigate aggregate formation and adhesion (< 20%). The cumulative output collected as a wet or dry aerosol is not aggregated to the same extent as the residual protein remaining in the nebulizer. Ultrasonic nebulization also results in aggregation, but the extent of denaturation is dependent upon the nebulizer used and is related to the heating of nebulizer solutions. Cooling of the nebulizer solution during operation (< 30 degrees C) minimizes aggregation (< 5%), and bioactivity is retained.