This study elucidates the importance of thermal reversibility as it pertains to the minimization of recombinant human Flt3 ligand aggregation and its potential role for determining solution conditions that can achieve the greatest long-term storage stability. Both thermal reversibility and Tm were evaluated as microcalorimetric parameters of stability within the range extending from pH 6 to 9, where the Tm was shown to plateau near 80 degrees C. Within this region, the reversibility was shown to decrease from 96. 6% to 15.2% while the pH was increased from 6 to 9, respectively. Accelerated stability studies conducted at 50 degrees C exhibited rates of aggregation augmented by pH that inversely correlated with the thermal reversibility data. Namely, high thermal reversibility at the Tm plateau correlated with slower rates of aggregation. Enthalpic calorimetric to van't Hoff ratios (DeltaH1/DeltaHv) yielded results close to unity within the plateau region, suggesting that the unfolding of rhFlt3 ligand was approximately two-state. Evidence that unfolding preceded the formation of the aggregate was provided by far-UV CD data of a soluble islolate of the aggregated product exhibiting a 28% loss of alpha-helix offset by a 31% gain in beta-sheet. This information combined with the thermal reversibility data provided compelling evidence that unfolding was a key event in the aggregation pathway at 50 degrees C. Minimization of aggregation was achieved at pH 6 and corroborated by evidence acquired from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size exclusion data. Correspondingly, the bioactivity was found to be optimal at pH 6. The findings link thermal reversibility to the propensity of Flt3 ligand to aggregate once unfolded in the Tm plateau region and provide a basis for relating the reversibility of thermal denaturation to the prediction of long-term storage stability in aqueous solution.