PTB-associated splicing factor (PSF) has been implicated in both early and late steps of pre-mRNA splicing, but its exact role in this process remains unclear. Here we show that PSF interacts with p54nrb, a highly related protein first identified based on cross-reactivity to antibodies against the yeast second-step splicing factor Prp18. We performed RNA-binding experiments to determine the preferred RNA-binding sequences for PSF and p54nrb, both individually and in combination. In all cases, iterative selection assays identified a purine-rich sequence located on the 3′ side of U5 snRNA stem 1b. Filter-binding assays and RNA affinity selection experiments demonstrated that PSF and p54nrb bind U5 snRNA with both the sequence and structure of stem 1b contributing to binding specificity. Sedimentation analyses show that both proteins associate with spliceosomes and with U4/U6.U5 tri-snPNP.