The methylation of histone H3 at lysine 4 (H3K4me) is critical for the formation of transcriptionally active chromatin in eukaryotes. In yeast, Drosophila, and some human cell lines, H3K4me is globally stimulated by the monoubiquitylation of histone H2B (H2Bub1), another histone modification associated with transcription. The mechanism of this "trans-histone" modification pathway remains uncertain, and studies carried out in different experimental systems have suggested that H2Bub1 could either influence the subunit composition of methyltransferase complexes or directly stimulate methyltransferase activity. We have reconstituted this pathway in vitro using the native H3K4-specific methyltransferase complex Set1C purified from the fission yeast Schizosaccharomyces pombe and chromatin substrates that contain semisynthetic H2Bub1. We found that the activity of S. pombe Set1C toward nucleosomal histone H3 is directly enhanced by H2Bub1 in vitro. Importantly, Set1C purified from cells lacking H2Bub1 retained activity on free histone substrates, suggesting that Set1C remains intact in the absence of H2Bub1. Chromatin immunoprecipitation assays revealed a defect in recruitment of intact Set1C to transcribed chromatin in H2Bub1-deficient mutants. Our data argue that trans-histone crosstalk in S. pombe involves direct enhancement of Set1C methyltransferase activity by H2Bub1 and suggest that this represents a conserved aspect of H2Bub1-H3K4me crosstalk in eukaryotes.