In order to produce correctly spliced messenger RNA, two catalytic splicing steps are required. After catalytic step I, a major remodeling of the spliceosome occurs to establish the active site for step II. During the second step of mRNA splicing, exon 1 attacks an adenine-guanine (AG) dinucleotide at the 3' splice site. SLU7, the human homolog of the yeast step II splice factor Slu7, is required for selection of the correct AG. Human SLU7 associates with the spliceosome late in the splicing pathway prior to recognition of the 3' splice site for step II. SLU7 depletion in HeLa nuclear extract reveals that SLU7 is required to hold exon 1 tightly within the spliceosome for attack on a prespecified AG.