Two forms of spliceosomes were found in higher eukaryotes. The major form contains the U1, U2, U4, U5, and U6 snRNAs; the minor form contains the U11, U12, U4atac, U5, and U6atac snRNAs. Assembly and function of the major form are based on a complex dynamic of UsnRNA–UsnRNA and UsnRNA–pre-mRNA interactions, and the involved UsnRNA segments are highly posttranscriptionally modified in plants and vertebrates. To further characterize the minor form of spliceosomes, we looked for the Ψ residues in HeLa cells' U11, U12, U4atac, and U6atac snRNAs, using chemical approaches. Four Ψ residues were detected in total for these four atac UsnRNAs, compared to 20 in their counterparts of the major spliceosomes. The two Ψ residues detected in U12 are also found in U2 snRNA. One of them belongs to the branch-site-recognition sequence. It forms one of the base pairs that bulge out the A residue, responsible for the nucleophilic attack. Conservation of this strategic Ψ residue probably reflects a functional role. Another Ψ residue was detected in a U4atac snRNA segment involved in formation of helix II with U6atac. The fourth one was detected in the additional stem-loop structure present at the 3′ end of U6atac snRNA. Differences in Ψ content of the atac and major UsnRNAs of human cells may participate in the differentiation of the two splicing systems. Based on secondary structure similarity, U2 and U12 snRNAs on the one hand and U4 and U4atac snRNAs on the other hand may share common Ψ synthases.