TBP-interacting protein 120 (TIP120) has been identified by TBP-mediated affinity screening. Classical TIP120, TIP120A, which functions as a transcriptional activator, is expressed ubiquitously whereas TIP120B is specifically expressed in muscle tissues. We found that TIP120B gene was induced in C2C12 myoblasts when these cells differentiated into myotubes, whereas TIP120A gene expression was down-regulated. Whole-mount in situ hybridization revealed that TIP120B mRNA was concentrated in limb buds of mouse embryos. TIP120B is thus thought to be a myogenesis-responding gene. We searched for TIP120B-binding proteins by yeast two-hybrid screening and identified NOT3. NOT3, a constituent of CCR4-NOT complex, is suggested to be involved in global gene regulation via interaction with TBP. The human NOT3 (hNOT3L), which we identified, has an extra 144 amino acids (AAs) at the C-terminus of a classical NOT3. GST pull-down and yeast two-hybrid assays demonstrated that hNOT3L is associated with TIP120B but not with TIP120A. A hNOT3L-specific C-terminal region of 92 AAs was assigned as a TIP120B-interacting domain. The N-terminus of 209 AAs of TIP120B was responsible for this binding. TIP120B presumably affects tissue-specific transcriptional regulation via interaction with NOT3.