The thermal stability of adenovirus type 5 (Ad5) was investigated over the pH range 3-8 employing a variety of biophysical techniques under conditions of low and high ionic strength. Analysis of the structural stability of Ad5 by dynamic light scattering, intrinsic and extrinsic fluorescence, and second derivative UV absorption spectroscopies suggest that the capsid stability of Ad5 increases with decreasing pH under both ionic strength conditions. Significant aggregation, however, was observed at pH < or = 5 under conditions of low ionic strength. These studies also suggest that the physical stability of Ad5 is significantly enhanced under acidic conditions in the presence of 1 M NaCl. Evaluation of the quaternary structural stability of Ad5 by dynamic light scattering and extrinsic fluorescence spectroscopy suggest that the Ad5 capsid undergoes a two-step dismantling process wherein the viral particles initially expand in size near 50 degrees C and the DNA core is at least partially exposed to the surrounding solvent. Complete capsid disassembly and total exposure of the DNA core follows at higher temperatures. Data generated during these studies were combined employing a multidimensional eigenvector approach that combines data from numerous techniques into a colored representation. This picture, or "empirical phase diagram," provides an intuitive representation of the physical stability of Ad5 over the pH range 4-8 from 10 degrees C to 85 degrees C.