α-Actinins are proteins that are involved in the formation of multidimensional structures of actin filaments. Their general structure consists of three domains: actin-binding domain, central rod domain and calmodulin-like domain. The physiologically active form is represented by antiparallel homodimers. In general, α-actinin-1 and α-actinin-4 are considered as different homodimeric entities, but recent studies have confirmed the existence of heterodimers in cancer cell lines. The purpose of our work was to evaluate the stability of the heterodimer by analyzing sequences and structural models, also in the aspect of phosphorylation of amino acid residues. Alignment results of the sequences α-actinin-2, α-actinin-1 and α-actinin-4 have shown a high percentage of amino acid identity (for isoform 1 78.26%, for isoform 4 75.00%). This allowed us to prepare a model of homodimer and heterodimer of isoforms 1 and 4 based on the structure of α-a ctinin-2 with a relatively high degree of reliability. The analysis of the interface in the 3D structural model of the heterodimer predicted the interface of 5167.8 Å2 and ΔiG = -28 kcal/mol, which represents the tendency for hydrophobic interactions. For its interface hydrophobic interactions were predicted and confirmed by the results of the InterProSurf server. Based on the data provided by InterProSurf and PDBePISA, we determine 94 interaction amino acids in the α-actinin-1 chain and 100 interaction amino acids in the α-actinin-4 chain. Based on results provided by NetPhos 3.1 server for the prediction of phosphorylation sites and databases of protein phosphorylation - qPhos and EPSD, we assume 4 (α-actinin-1) and 6 (α-actinin-4) phosphorylation sites, respectively, located on the interface surface of the subunits and as such could potentially affect the stability of the heterodimer.