Human splicing factors Hprp3p and Hprp4p are associated with the U4/U6 small nuclear ribonucleoprotein particle, which is essential for the assembly of an active spliceosome. Currently, little is known about the specific roles of these factors in splicing. In this study, we characterized the molecular interaction between Hprp3p and Hprp4p. Constructs were created for expression of Hprp3p or its mutants in bacterial or mammalian cells. We showed that antibodies against either Hprp3p or Hprp4p were able to pull-down the Hprp3p-Hprp4p complex formed in Escherichia coli lysates. By co-immunoprecipitation and isothermal titration calorimetry, we demonstrated that purified Hprp3p and its mutants containing the central region, but lacking either the N-terminal 194 amino acids or the C-terminal 240 amino acids, were able to interact with Hprp4p. Conversely, Hprp3p mutants containing only the N- or C-terminal region did not interact with Hprp4p. In addition, by co-immunoprecipitation, we showed that intact Hprp3p and its mutants containing the central region interacted with Hprp4p in HeLa cell nuclear extracts. Primer extension analysis illustrated that the central region of Hprp3p is required to maintain the association of Hprp3p-Hprp4p with U4/U6 small nuclear RNAs, suggesting that this Hprp3p/Hprp4p interaction allows the recruitment of Hprp4p, and perhaps other protein(s), to the U4/U6 small nuclear ribonucleoprotein particle.