S100 proteins form characteristic homo- and/or heterodimers that play a role in calcium-mediated signaling. We characterized the formation of the human S100A8/S100A9 heterodimer using the yeast two-hybrid system. Employing site-directed mutagenesis we found that distinct hydrophobic amino acids of helix I/I′ are located at a crucial site of the S100A8/S100A9 dimer interface, whereas conserved residues within helix IV/IV′ are not important for heterodimerization. Furthermore, amino acids Y16 and F68 prevent homodimerization of human S100A8. These data demonstrate for the first time the functional relevance of distinct hydrophobic amino acids for human S100A8/S100A9 complex formation in vivo.