Methylation on lysine 4 of histone H3 (H3K4) is one of the prominent histone modification marks that correlate strongly with active transcription in yeast and metazoans. Accumulating studies in metazoans have implicated misregulation of H3K4 methylation in the pathogenesis of cancer and in developmental defects, further emphasizing the importance of understanding the regulation of H3K4 methylation. Yeast genetic studies have documented a crosstalk between H2B ubiquitylation (H2Bub) and H3K4 methylation, but little direct biochemical evidence exists explaining the mechanism underlying H3K4 methylation on chromatin templates for the human H3K4 methyltransferase complexes. By taking advantage of an in vitro histone methyltransferase assay employing purified human H3K4 methyltransferase complexes and recombinant chromatin templates, we characterize biochemical properties of human H3K4 methyltransferase complexes. These studies establish minimal components of human H3K4 methyltransferase complexes required for H3K4 methylation and also provide a mechanistic basis for H3K4 methylation in mammalian cells.