The conformation of Saccharomyces cerevisiae U3 snRNA (snR17A RNA) in solution was studied using enzymatic and chemical probes. In vitro synthesized and authentic snR17A RNAs have a similar conformation in solution. The S. cerevisiae U3 snRNA is folded in two distinct domains. The 5'-domain has a low degree of compactness; it is constituted of two stem-loop structures separated by a single-stranded segment, which has recently been proposed to basepair with the 5'-ETS of pre-ribosomal RNA. We demonstrate that, as previously proposed, the 5'-terminal region of U3 snRNA has a different structure in higher and lower eukaryotes and that this may be related to pre-rRNA 5'-ETS evolution. The S. cerevisiae U3 snRNA 3'-domain has a cruciform secondary structure and a compact conformation resulting from an higher order structure involving the single-stranded segments at the center of the cross and the bottom parts of helices. Compared to tRNA, where long range interactions take place between terminal loops, this represents another kind of tertiary folding of RNA molecules that will deserve further investigation, especially since the implicated single-strands have highly evolutionarily conserved primary structures that are involved in snRNP protein binding.