Methylation on lysine 4 of histone H3 (H3K4) is one of the prominent histone modification marks that correlate strongly with active transcription in eukaryotes. 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. In budding yeast, a single H3K4 methyltransferase (Set1) complex can methylate all H3K4 in an H2B ubiquitylation-dependent manner. However, presence of at least six H3K4 methyltransferase (SET1A, SET1B, MLL1, MLL2, MLL3 and MLL4) complexes in mammalian cells has complicated functional characterization of each complex because of their possible redundant and non-redundant roles. To understand the roles of H3K4 methyltransferases in transcriptional regulation in mammalian cells, we have deployed biochemical analyses with purified human H3K4 methyltransferase complexes. By taking advantage of an in vitro histone methyltransferarse assay employing a reconstituted human H3K4 methyltransferase complexes and a recombinant chromatin template containing fully ubiquitylated H2B, we found that a subset of human H3K4 methyltransferase complexes exhibit H2B ubiquitylation-dependent H3K4 methylation activity. In addition, we demonstrate different subunit compositions and subunit interaction networks in human H3K4 methyltransferase complexes. Our studies establish minimal components of the H3K4 methyltransferase complexes required for H3K4 methylation and provide a mechanistic basis for H3K4 methylation in mammalian cells.