β-Thymosins are actin monomer-binding polypeptides that are expressed in a neuronal growth-specific manner during embryonic development. Here, we show that regenerating retinal ganglion cells and non-neuronal cells of the optic nerve transiently activate β-thymosin transcription after optic nerve lesion in the zebrafish. In retinal cell cultures, β-thymosin is found at highest concentration in growth cones, branching points and varicosities of neurite-extending retinal ganglion cells. These places often exhibit reduced phalloidin staining, indicating that β-thymosin promotes the disassembly of actin filaments. β-Thymosin distribution within neurons in culture is distinct from actin, tubulin and the actin-severing protein gelsolin. Ectopic expression of β-thymosin in a central nervous system (CNS) catecholaminergic cell line leads to alterations in the shape of the cell bodies and neurites. β-Thymosin-positive cells spread more fully and exhibit an excessive degree of branching. We partially cloned two other actin-binding proteins, profilin and gelsolin, and analysed their expression patterns. Profilin is constitutively expressed in virtually all cells. Gelsolin, like β-thymosin, is selectively increased in regenerating retinal ganglion cells. During development, however, gelsolin mRNA is not detected in the nervous system. These findings indicate that distinct mechanisms control the actin cytoskeleton in embryonic and regenerating neurons, and that β-thymosin may be a major regulator of actin dynamics in the zebrafish CNS.