The thymosin β s (Tβ s) are polypeptide regulators of actin dynamics that are critical for the growth and branching of neurites in developing neurons. We found that mRNAs for Tβ 4, Tβ 10, and Tβ 15 were highly expressed in the developing rat brain during neuritogenesis, supporting a role for the Tβ s in this process. Overexpression of the Tβ s increased the number of neurite branches per neuron in cultured hippocampal and cerebral cortex neurons, and Tβ 15 had the greatest effect. Actin binding activity appears to be essential for the branch-promoting activity of Tβ s because two mutants of To 15 lacking monomeric actin binding activity failed to stimulate branch formation. We also found that transfection of siRNA against Tβ 15 reduced branching. Taken together, these data suggest that the three Tβ s, and especially Tβ 15, stimulate neurite branching during brain development. © 2005 Elsevier Inc. All rights reserved.