In the present work, we have analyzed the expression and subcellular localization of all the members of inositide-specific phospholipase C (PLCβ) family in muscle differentiation, given that nuclear PLCβ1 has been shown to be related to the differentiative process. Cell cultures of C2C12 myoblasts were induced to differentiate towards the phenotype of myotubes, which are also indicated as differentiated C2C12 cells. By means of immunochemical and immunocytochemical analysis, the expression and subcellular localization of PLCβ1, β2, β3, β4 have been assessed. As further characterization, we investigated the localization of PLCβ isoenzymes in C2C12 cells by fusing their cDNA to enhanced green fluorescent protein (GFP). In myoblast culture, PLCβ4 was the most expressed isoform in the cytoplasm, whereas PLCβ1 and β3 exhibited a lesser expression in this cell compartment. In nuclei of differentiated myotube culture, PLCβ1 isoform was expressed at the highest extent. A marked decrease of PLCβ4 expression in the cytoplasm of differentiated C2C12 cells was detected as compared to myoblasts. No relevant differences were evidenced as regards the expression of PLCβ3 at both cytoplasmatic and nuclear level, whilst PLCβ2 expression was almost undetactable. Therefore, we propose that the different subcellular expression of these PLC isoforms, namely the increase of nuclear PLCβ1 and the decrease of cytoplasmatic PLCβ4, during the establishment of myotube differentiation, is related to a spatial-temporal signaling event, involved in myogenic differentiation. Once again the subcellular localization appears to be a key step for the diverse signaling activity of PLCβs. © 2004 Wiley-Liss, Inc.