The demonstration that AcSDKP, a new regulator of the hematopoietic system, is formed in the bone marrow by a one-step enzymatic maturation processing of thymosin beta 4 (T beta 4) is presented. AcSDKP and T beta 4 were both detected in bone marrow cells (BMC). Incubation of [3H]T beta 4 with either intact or lysed BMC led to the formation of [3H]AcSDKP, whereas the labeled tetrapeptide was not degraded under these conditions. Model enzymatic degradation of T beta 4 carried out with bacterial enzymes suggests that a mammalian endoproteinase Asp-N might be involved in the formation of AcSDKP through the specific cleavage of the Pro4-Asp5 peptidic bond of T beta 4. In contrast, alpha-prolyl-endopeptidase was ineffective in carrying out a similar processing.