To examine the relative role of halogen bonding and hydrophobic interactions in the inhibition of human CK2 alpha by 4,5,6,7-tetrabromobenzotriazole (TBBt), we have synthesized a series of 5-substituted benzotriazoles (Bt) and the corresponding 5-substituted 4,6,7-tribromobenzotriazoles (Br(3)Bt) and examined their inhibition of human CK2 alpha relative to that of TBBt. The various C(5) substituents differ in size (H and CH3), electronegativity (NH2 and NO2), and hydrophobicity (COOH and Cl). Some substituents were halogen bond donors (Cl, Br), while others were fluorine bond donors (F and CF3). Most of the 5-substituted analogues of Br(3)Bt (with the exception of COOH and NH2) exhibited inhibitory activity comparable to that of TBBt, whereas the 5-substituted analogues of the parent Bt were only weakly active (Br, Cl, NO2, CF3) or inactive. The observed effect of the volume of a ligand molecule pointed to its predominant role in inhibitory activity, indicating that presumed halogen bonding, identified in crystal structures and by molecular modeling, is dominated by hydrophobic interactions. Extended QSAR analysis additionally pointed to the monoanion and a preference for the N(1)-H protomer of the neutral ligand as parameters crucial for prediction of inhibitory activity. This suggests that the monoanions of TBBt and its congeners are the active forms that efficiently bind to CK2 alpha, and the binding affinity is coupled with protomeric equilibrium of the neutral ligand.