dHAND and eHAND are basic helix-loop-helix (bHLH) transcription factors expressed during embryogenesis and are required for the proper development of cardiac and extraembryonic tissues. HAND genes, like the myogenic bHLH genes, are classified as class B bHLH genes, which are expressed in a tissue-restricted pattern and function by forming heterodimers with class A bHLH proteins. Myogenic bHLH genes are shown not to form homodimers efficiently, suggesting that their activity is dependent on their E-protein partners. To identify HIPs (HAND-interacting proteins) that regulate the activity of the HAND genes, we screened an 9.5-10.5-day-old mouse embryonic yeast two-hybrid library with eHAND. Several HIPs held high sequence identity to eHAND, indicating that eHAND could form and function as a homodimer. Based on the high degree of amino acid identity between eHAND and dHAND, it is possible that dHAND could also form homodimers and heterodimers with eHAND. We show using yeast and mammalian two-hybrid assays as well as biochemical pull-down assays that eHAND and dHAND are capable of forming both HAND homo- and heterodimers in vivo. To investigate whether HAND genes form heterodimers with other biologically relevant bHLH proteins, we tested and show HAND heterodimerization with the recently identified Hairy-related transcription factors, HRT1-3. This finding is exciting, because both HRT and HAND genes are coexpressed in the developing heart and limb and both have been implicated in establishing tissue boundaries and pattern formation. Moreover, competition gel shift analysis demonstrates that dHAND and eHAND can negatively regulate the DNA binding of MyoD/E12 heterodimers in a manner similar to MISTI and Id proteins, suggesting a possible transcriptional inhibitory role for HAND genes. Taken together, these results show that dHAND and eHAND can form homo- and heterodimer combinations with multiple bHLH partners and that this broad dimerization profile reflects the mechanisms by which HAND genes regulate transcription.