Thymosin beta 4 (T beta 4) is a 43-amino acid signature motif peptide that defines the betathymosin (beta T) family of proteins. beta Ts are intrinsically unstructured in their free states and undergo disorder-to-order transitions in carrying out their biological functions. This property poses challenges in determining their 3D structures, mainly favoring structural studies on the complexes formed between beta Ts and their interaction partners. One of the beta Ts' primary binding partners is monomeric actin, a major component of the cytoskeleton in eukaryotic cells. T beta 4' s role in this system is to maintain the highly concentrated pool of monomeric actin that can be accessed through profilin by actin filament nucleating machineries. Here, we give an account of the structures of beta Ts that have been illuminated by nuclear magnetic resonance (NMR) and X-ray crystallography. NMR has been the method of choice for probing regions that have intrinsic conformational preference within the largely disordered beta Ts in their native states in solution. X-ray crystallography has demonstrated at atomic detail how beta Ts interact with actin. Detailed analysis of these structures highlights the disorder-to-order transition of T beta 4 in binding to actin and its isoform specificity.