THE hydrolysis of ATP by a group of RNA dependent ATPases (DEAD/H proteins(1)) is required for spliceosome assembly, but not for the subsequent transesterification reactions(2). Little is known about the function of these ATPases in relation to the RNA conformational changes that occur in formation of active structures, in which U2/U6 small nuclear RNA (snRNA) interactions(3,4) are essential for splicing to take place, Using a synthetic lethal genetic screen, we have isolated four yeast splicing factors involved in U2/U6 snRNA interactions (D.X. et al., manuscript in preparation). The RNA-dependent ATPase activity associated with one such factor, the Slt22 protein, is stimulated preferentially by annealed U2/U6 snRNAs. Both mutant slt22-1 and U2 snRNA cause a reduction in stimulation, The slt22-1 mutation blocks splicing at or before the first step, resulting in the accumulation of an unusual complex which lacks U5 snRNA. Our results indicate that the U2/U6 snRNA interactions facilitated by Slt22 are also Involved in the interaction of U5 snRNA with tire spliceosome.