Histone side chains are post-translationally modified at multiple sites, including at Lys36 on histone H3 (H3K36). Several enzymes from yeast and humans, including the methyltransferases SET domain-containing 2 (Set2) and nuclear receptor SET domain-containing 1 (NSD1), respectively, alter the methylation status of H3K36, and significant progress has been made in understanding how they affect chromatin structure and function. Although H3K36 methylation is most commonly associated with the transcription of active euchromatin, it has also been implicated in diverse processes, including alternative splicing, dosage compensation and transcriptional repression, as well as DNA repair and recombination. Disrupted placement of methylated H3K36 within the chromatin landscape can lead to a range of human diseases, underscoring the importance of this modification.