U4 small nuclear RNA (snRNA) plays a fundamental role in the process of premessenger RNA splicing, yet many questions remain regarding the location, interactions, and roles of its functional domains. To address some of these questions, we developed the first in vitro reconstitution system for yeast U4 small nuclear ribonucleoproteins (snRNPs). We used this system to examine the functional domains of U4 by measuring reconstitution of splicing, U4/U6 base-pairing, and triple-snRNP formation. In contrast to previous work in human extracts and Xenopus oocytes, we found that the 39 stem-loop of U4 is necessary for efficient base-pairing with U6. In particular, the loop is sensitive to changes in both length and sequence. Intriguingly, a number of mutations that we tested resulted in more stable interactions with U6 than wild-type U4. Nevertheless, each of these mutants was impaired in its ability to support splicing, indicating that these regions of U4 have functions subsequent to base pair formation with U6. Our data suggest that one such function is likely to be in tri-snRNP formation, when U5 joins the U4/U6 di-snRNP. We have identified two regions, the upper stem of the 39 stem-loop and the central domain, that promote tri-snRNP formation. In addition, the loop of the 39 stem-loop promotes di-snRNP formation, while the central domain and the 39-terminal domain appear to antagonize di-snRNP formation.