A mutation (U4-G14C) that destabilizes the base-pairing interaction between U4 and U6 snRNAs causes the accumulation of a novel complex containing U4, U6 and Prp24, a protein with RNA binding motifs. An analysis of suppressors of this cold-sensitive mutant led to the hypothesis that this complex is normally a transient intermediate in the annealing of U4 and U6. It was proposed that Prp24 must be released to form a fully base-paired U4/U6 snRNP. By using a chemical probing method we have tested the prediction that nucleotides A40-C43 in U6 mediate the binding of Prp24. Consistent with the location of recessive suppressors in U6, we find that residues A40-C43 are protected from chemical modification in U4/U6 complexes from the U4-G14C mutant but not from the wild-type or suppressor strains carrying mutations in U6 or PRP24. Furthermore, we find that base-pairing is substantially disrupted in the mutant complexes. Notably, the base-paired structure is restored in recessive suppressors despite the presence of a mismatched base-pair at the U4-G14C site. Our results support the model that Prp24 binds to U6 to promote its association with U4, but must dissociate to allow complete annealing.