The neurohypophyseal nonapeptide Arg8 vasopressin (AVP) promotes differentiation of cultured L6 and L5 myogenic cell lines and mouse primary satellite cells. Here, we investigated the molecular mechanism involved in the induction of the myogenic program by AVP. In L6 cells, AVP treatment rapidly induces Myf-5, myogenin, and myocyte enhancer factor 2 (MEF2) mRNAs, without affecting the expression of known myogenic growth factors such as IGF-I, IGF-II, or their receptors. In the presence of cycloheximide, AVP up-regulates the expression of MEF2, but not of myogenin, indicating that the synthesis of a protein intermediate is not necessary for MEF2 induction. Notably, AVP treatment activates a calcium/calmodulin kinase signaling pathway that induces cytosolic compartmentalization of the histone deacetylase 4, a mechanism related to the transcriptional activation of MEF2. The activity of chloramphenicol acetyltransferase reporter constructs carrying the Myo184 and Myo84 fragments of the myogenin promoter is also induced by AVP. Mutation of the MEF2 site completely abolishes the response to AVP, whereas deletion of the E1 site present in pMyo84 does not impair this response. Together, these results show that AVP induces myogenic differentiation through the transcriptional activation of MEF2, a mechanism that is critical for myogenesis.