Here we report further characterization of an in vitro assay system for exon ligation by the human spliceosome in which the 3′ splice site AG is supplied by a different RNA molecule than that containing the 5′ splice and branch sites. By varying the time during splicing reactions when the 3′ splice site AG is made available to the splicing machinery, we show that AG recognition need not occur until after lariat formation. Thus an early AG recognition event required for spliceosome formation and lariat formation on some mammalian introns is not required for exon ligation. Depletion/add-back studies and cold competitor challenge experiments reveal that commitment of a 3′ splice site AG to exon ligation requires NTP hydrolysis. Because it both physically and kinetically uncouples exon ligation from spliceosome assembly and lariat formation, the bimolecular system will be a valuable tool for further mechanistic analysis of the second step of splicing.