Histone H3 lysine 36 methylation is a conserved histone modification that is critical for maintaining eukaryotic transcriptional fidelity and genomic stability. In yeast, this modification is catalyzed by Set2, an ortholog of the mammalian H3K36 methyltransferase SETD2. Previous genetic, biochemical, and structural studies showed that Set2 activity is repressed by a Set2 autoinhibitory domain (AID) and that activation requires the direct binding of the histone chaperone Spt6. To study the role of Spt6 and Set2 autoinhibition in vivo, we have isolated and analyzed multiple classes of Spt6 and Set2 mutants. Our results suggest an autoinhibited form of Set2 in which the catalytic domain is bound by the AID. In strong agreement with our genetic results, biophysical experiments demonstrate that the Set2 catalytic domain and Set2 AID physically interact, and that the autoinhibition mutants abolish this interaction. The hydrogen deuterium exchange was used to study effect of mutations on the protein structure.