Thymosin beta 4 (Tbeta4) is a peptide of 43 amino acids, mainly recognized as a regulator of actin polymerization by sequestering G-actin. Meanwhile, the peptide has been implicated in lymphocyte maturation, carcinogenesis, apoptosis, angiogenesis, blood coagulation and wound healing. The peptide is also involved in lesion-induced neuroplasticity through microglia upregulation and it participates in the growth of neuronal processes. However, its precise cellular localization throughout the entire body of the mouse has not been documented. We therefore initiated a detailed investigation of the tissue distribution and cellular expression of the Tbeta4 peptide and its precursor mRNA by immunocytochemistry and in situ hybridization, respectively. In the brain, Tbeta4 was clearly present in neurons of the olfactory bulb, neocortex, hippocampus, striatum, amygdala, piriform cortex and cerebellum, and in microglia across the entire brain. We further localized Tbeta4 in cells, typically with many processes, inside thymus, spleen, lung, kidney, liver, adrenal gland, stomach and intestine. Remarkably, Tbeta4 was thus associated with microglia and macrophages, the differentiated phagocytic cells residing in every tissue. Motility and phagocytosis, two important activities of macrophages, depend on actin, which can explain the presence of Tbeta4 in these cells.