Recently we identified a new family of histone deacetylases in higher eukaryotes related to yeast HDA1 and showed their differentiation-dependent expression. Data presented here indicate that HDAC5 (previously named mHDA1), one member of this family, might be a potent regulator of cell differentiation by interacting specifically with determinant transcription factors. We found that HDAC5 was able to interact in vivo and in vitro with MEF2A, a MADS box transcription factor, and to strongly inhibit its transcriptional activity. Surprisingly, this repression was independent of HDAC5 deacetylase domain. The N-terminal non-deacetylase domain of HDAC5 was able to ensure an efficient repression of MEF2A-dependent transcription. We then mapped protein domains involved in the HDAC5-MEF2A interaction and showed that MADS box/MEF2-domain region of MEF2A interacts specifically with a limited region in the N-terminal part of HDAC5 which also possesses a distinct repressor domain. These data show that two independent class II histone deacetylases HDAC4 and HDAC5 are able to interact with members of the MEF2 transcription factor family and regulate their transcriptional activity, thus suggesting a critical role for these deacetylases in the control of cell proliferation/differentiation. Acetylation of chromatin proteins and transcription factors is part of a complex signaling system that is largely involved in the control of gene expression (1). Thus far, the specific involvement of histone acetyltransferases and deacetylases in the control of individual gene expression has been clearly established (2, 3). One major role of these enzymes is the control of cell differentiation in response to specific signals. Evidence exists for the participation of the RPD3-related members in this process (4). Recently, however, a new family of higher eukaryotic histone deacetylases, distinct from the already characterized RPD3-related members, has been identified (5-7). These enzymes are related to yeast HDA1 histone deacetylase and within the cloned members, two show sequence homology and the same domain organization and are called HDAC4 and HDAC5 (5, 6). Despite this homology, HDAC4 and HDAC5 are probably capable of exerting distinct functions, since immunoprecipitation experiments showed that in cells they can be associated with different partners (6). A member, named mHDA2/HDAC6, shows unique features within deacetylases, in that it possesses two HDA1 homology domains (5, 6). In contrast to the RPD3-related members, the expression of these genes is not ubiquitous. HDAC5 and mHDA2/HDAC6 expression is activated upon cell differentiation (5). These observations suggest that members of the class II histone deacetylases may play a specific role in the regulation of cell differentiation. Looking for potential partners of HDAC5, we obtained evidence of interaction between HDAC5 and MEF2 transcription factors. We therefore focused our efforts on investigating this issue. The MEF2 family of transcription factors belongs to the large family of MADS...