The formation of pseudouridine (psi) in human U1, U2 and U5 small nuclear RNAs (snRNAs) was investigated using HeLa cell extracts. Unmodified snRNAs were synthesized in vitro and the extent of psi formation was determined after incubation in cell extracts. The formation of psi on labelled substrates was monitored in the presence of 5-fluorouracil (5-FU)-containing snRNAs as inhibitors of psi formation. The conversion of uridine to psi was inhibited only when the cognate 5-FU-containing inhibitor snRNA was included in the reaction. For example, 5-FU-containing U1 RNA inhibited psi formation in unmodified U1 RNA, but not in (unmodified) U2 or U5 RNAs. The results suggest that there are at least three activities that form psi in these snRNAs. The 5-FU-containing RNAs were stable during incubation in the cell extracts. A 12-fold molar excess of unlabelled U1 RNA did not inhibit psi formation on a labelled U1 RNA substrate, whereas a 3-fold molar excess of 5-FU-containing U1 RNA nearly abolished psi formation on the U1 substrate. The fact that 5-FU-containing snRNAs are potent inhibitors of psi formation in these pre-mRNA splicing cofactors raises the possibility that this is related to the cytotoxicity of fluoropyrimidines in cancer chemotherapy.