The head of the P22 bacteriophage is interrupted by a unique dodecameric portal vertex that serves as a conduit for the entrance and exit of the DNA. Here, the in vitro unfolding/refolding processes of the portal protein of P22 were investigated at different temperatures (1, 25, and 37°C) through the use of urea and high hydrostatic pressure (HHP) combined with spectroscopic techniques. We have characterized an intermediate species, IU, which forms at 25°C during unfolding or refolding of the portal protein in 2-4 M urea. IU readily forms amorphous aggregates, rendering the folding process irreversible. On the other hand, at 1°C, a two-state process is observed (ΔGf = -2.2 kcal/mol). When subjected to HHP at 25 or 37°C, the portal monomer undergoes partial denaturation, also forming an intermediate species, which we call IP. IP also tends to aggregate but, differently from IU, aggregates into a ring-like structure as seen by size-exclusion chromatography and electron microscopy. Again, at 1°C the unfolding induced by HHP proved to be reversible, with ΔGf = -2.4 kcal/mol and ΔV = 72 mL/mol. Interestingly, at 25 °C, the binding of the hydrophobic probe bis-ANS to the native portal protein destabilizes it and completely blocks its aggregation under HHP. These data are relevant to the process by which the portal protein assembles into dodecamers in vivo, since species such as IP must prevail over I U in order to guarantee the proper ring formation. © 2007 American Chemical Society.