The strong specific binding of streptavidin (SA) to biotin is utilized in numerous biotechnological applications. The SA tetramer is also known to exhibit significant stability, even in the presence of sodium dodecylsulfate (SDS). Despite its importance, relatively little is known about the nature of the thermal denaturation pathway for SA. This work uses a homogeneous SA preparation to expand on the data of previous literature reports, leading to the proposal of a model for temperature induced structural changes in SA. Temperature dependent data were obtained by SDS and native polyacrylamide gel electrophoresis (PAGE), differential scanning calorimetry (DSC), and fluorescence and ultraviolet (UV)-visible spectroscopy in the presence and absence of SDS. In addition to the development of this model, it is found that the major thermal transition of SA in 1% SDS is reversible. Finally, although SA exhibits significant precipitation at elevated temperatures in aqueous solution, inclusion of SDS acts to prevent SA aggregation.