Thymosin β4 (Tβ4) is a multifunctional protein that has pleiotropic activities both intracellularly and extracellularly. The mechanisms by which it influences cellular processes such as adhesion, migration, differentiation, or apoptosis are not yet understood. Calcium is a ubiquitous signal molecule that is involved in the regulation of almost all cellular functions. Our data indicate that the release of Ca(2+) from intracellular stores following stimulation of cells with Tβ4 does not occur. Interestingly, Tβ4 becomes rapidly internalized, supporting the concept that it may express its activities via intracellular receptors.