MCAT elements and its cognate binding partners, the transcription enhancer factors (TEFs) play important roles in the regulation of expression of several muscle-specific genes. The biological effects of TEFs strongly depend on different co-factors, which might act as co-activators or anti-repressors to enable transcriptional activation of target genes by TEFs. Previously, we have cloned and characterized VITO-1, which acts as a skeletal muscle-specific transcriptional co-activator of TEFs. Here we describe the cloning and expression profile of a related gene, VITO-2 (also termed Vgl-3), which shares a high homology with VITO-1 in the SID domain responsible for interaction with TEFs. During early embryonic and fetal development VITO-2 is mainly expressed in the myogenic lineage with an onset of expression in the myotomes of somites VI at E9.5 slightly later than VITO-1. At later developmental stages VITO-2 is predominantly found in the nervous system. In adult mice VITO-2 was detected in different tissues, including skeletal muscle, heart, kidney, liver and brain, where it was found in cortical and cerebellar neurons as well as in Purkinje cells. The expression of VITO-2 in the mesoderm was repressed by the notch/delta pathway and activated by Myf-5 since Dll-1 mutant showed an aberrant expression of VITO-2 but not VITO-1 in the tail bud and in the caudal neural tube at E10.5 while Myf-5 mutant mice lack expression of VITO-1 and VITO-2 in somites until E10.5.