Integrins are a diverse family of adhesion molecules that mediate cell-cell and cell-matrix interactions. Of these, α_IIbβ₃ has been most extensively characterized, and provides a model for regulation of integrin function. A central feature of integrin regulation is transmembrane (TM) domain interaction: when integrins are inactive, the TM domains of their α and β subunits interact, but these domains separate when the integrin assumes its active conformation. Previously, we observed that the α_IIb and β₃ TM domains undergo both heteromeric and homomeric interactions and there is overlap of the helical interface that mediates these interactions. Less is known about whether other platelet integrin TM domains interact. To address this question, we examined the role of TM domain interactions in β₁ integrins, particularly α₂β₁ and α₅β₁. Using a novel ToxR-based assay for TM domain interactions in E. coli coupled with scanning mutagenesis, we identified residues important for homo- and heterooligomerization of the TM domains for each α/β chain, particularly small, polar residues that lie along one face of the TM helix. We also find overlap between the homo- and heterooligomeric interfaces for each of the integrin TM domains. We then determined the effect these residues have on the function of the full-length integrins by introducing several of the mutations into Jurkat and HEK293 suspension cell lines. Several of the TM domain mutations identified as important from the ToxR-assay caused activation of the full-length integrin, indicating these residues may interact in the inactive state. Overall, our results point to the essential role of TM regions in regulating integrin function and demonstrate the importance of small, polar residues in maintaining these integrins in their inactive conformations.