All pseudouridines identified in RNA are considered constitutive modifications. Here, we demonstrate that pseudouridylation of Saccharomyces cerevisiae U2 snRNA can be conditionally induced. While only Ψ35, Ψ42 and Ψ44 are detected in U2 under normal conditions, nutrient deprivation leads to additional pseudouridylation at positions 56 and 93. Pseudouridylation at position 56 can also be induced by heat shock. Detailed analyses have shown that Pus7p, a single polypeptide pseudouridylase known to modify U2 at position 35 and tRNA at position 13, catalyses Ψ56 formation, and that snR81 RNP, a box H/ACA RNP known to modify U2 snRNA at position 42 and 25S rRNA at position 1051, catalyses Ψ93 formation. Using mutagenesis, we have demonstrated that the inducibility can be attributed to the imperfect substrate sequences. By introducing Ψ93 into log-phase cells, we further show that Ψ93 has a role in pre-mRNA splicing. Our results thus demonstrate for the first time that pseudouridylation of RNA can be induced at sites of imperfect sequences, and that Pus7p and snR81 RNP can catalyse both constitutive and inducible pseudouridylation.