HBO1, an H4-specific histone acetylase, is a coactivator of the DNA replication licensing factor Cdt1. HBO1 acetylase activity is required for licensing, because a histone acetylase (HAT)-defective mutant of HBO1 bound at origins is unable to load the MCM complex. H4 acetylation at origins is cell-cycle regulated, with maximal activity at the G1/S transition, and coexpression of HBO1 and Jade-1 increases histone acetylation and MCM complex loading. Overexpression of the Set8 histone H4 tail-binding domain specifically inhibits MCM loading, suggesting that histones are a physiologically relevant target for licensing. Lastly, Geminin inhibits HBO1 acetylase activity in the context of a Cdt1-HBO1 complex, and it associates with origins and inhibits H4 acetylation and licensing in vivo. Thus, H4 acetylation at origins by HBO1 is critical for replication licensing by Cdt1, and negative regulation of licensing by Geminin is likely to involve inhibition of HBO1 histone acetylase activity.