The interaction of the U5-specific polypeptides with U5 snRNA was investigated by comparison of the differential accessibility towards nucleases and dimethylsulfate of defined regions of U5 snRNA in purified 20S and 10S U5 snRNPs. While 20S U5 snRNPs contain eight U5-specific proteins in addition to the common proteins, the 10S U5 snRNPs contain only the latter proteins. The results indicate that only the central part of stem/loop I of U5 snRNA including internal loops IL2 and IL2', contains binding sites for U5-specific proteins, suggesting that several U5-specific proteins may be bound to U5 snRNP via protein-protein interactions. Moreover, they show that the core polypeptides do not interact with stem/loop I.