The Z alpha domains of human ADAR1 (Z alpha(ADAR-1)) bind to Z-DNA via interaction mediated by the alpha 3-core and beta-hairpin. Five residues in the alpha 3 helix and four residues in the beta-hairpin play important roles in Z alpha function, forming direct or water-mediated hydrogen bonds with DNA backbone phosphates or interacting hydrophobically with DNA bases. To understand the roles of these residues during B-Z transition of duplex DNA, we performed NMR experiments on complexes of various Z alpha(ADAR1) mutants with a 6-bp DNA duplex at various protein-to-DNA molar ratios. Our study suggests that single mutations at residues K169, N173, or Y177 cause unusual conformational changes in the hydrophobic faces of helices alpha 1, alpha 2, and alpha 3, which dramatically decrease the Z-DNA binding affinity. ID imino proton spectra and chemical shift perturbation showed that single mutations at residues K170, R174, T191, P192, P193, or W195 slightly affected the Z-DNA binding affinity. A hydrogen exchange study proved that the K170A- and R174A-Z alpha(ADAR1) proteins could efficiently change B-DNA to left-handed Z-DNA via an active B-Z transition pathway, whereas the G2.C5 base pair was significantly destabilized compared to wild-type Z alpha(ADAR1).