Nuclear RNA splicing occurs in an RNA-protein complex, termed the spliceosome. U4/U6 snRNP is one of four essential small nuclear ribonucleoprotein (snRNP) particles (U1, U2, U5 and U4/U6) present in the spliceosome. U4/U6 snRNP contains two snRNAs (U4 and U6) and a number of proteins. We report here the identification and characterization of two human genes encoding U4/U6-associated splicing factors, Hprp3p and Hprp4p, respectively. Hprp3p is a 77 kDa protein, which is homologous to the Saccharomyces cerevisiae splicing factor Prp3p. Amino acid sequence analysis revealed two putative homologues in Caenorhabditis elegans and Schizosaccharomyces pombe. Polyclonal antibodies against Hprp3p were generated with His-tagged Hprp3p over-produced in Escherichia coli . This splicing factor can co-immunoprecipitate with U4, U6 and U5 snRNAs, suggesting that it is present in the U4/U6.U5 tri-snRNP. Hprp4p is a 58 kDa protein homologous to yeast splicing factor Prp4p. Like yeast Prp4p, the human homologue contains repeats homologous to the beta-subunit of G-proteins. These repeats are called WD repeats because there is a highly conserved dipeptide of tryptophan and aspartic acid present at the end of each repeat. The primary amino acid sequence homology between human Hprp4p and yeast Prp4p led to the discovery of two additional WD repeats in yeast Prp4p. Structural homology between these human and yeast splicing factors and the beta-subunit of G-proteins has been identified by sequence-similarity comparison and analysis of the protein folding by threading. Structural models of Hprp4p and Prp4p with a seven-blade beta-propeller topology have been generated based on the structure of beta-transducin. Hprp3p and Hprp4p have been shown to interact with each other and the first 100 amino acids of Hprp3p are not essential for this interaction. These experiments suggest that both Hprp3p and Hprp4p are components of human spliceosomes.