Splicing of pre-mRNAs involves two sequential transesterification reactions commonly referred to as the first and second steps. In Saccharomyces cerevisiae, four proteins, Prp16p, Prp17p, Prp18p, and Slu7p are exclusively required for the second step of splicing. The human homologs of Prp16p, Prp17p, and Prp18p have been identified, and the human proteins hPrp16 and hPrp18 have been shown to be required for the second step of splicing in vitro. Here we provide further evidence for the functional conservation of the second step factors between yeast and humans. Human hPrp17, which is 35% identical to the S. cerevisiae protein, is able to partially rescue the temperature-sensitive phenotype in a yeast strain where PRP17 has been knocked out, suggesting that the human and yeast proteins are functionally conserved. Overexpression of hPrp17 in the knockout yeast strain partially rescues the splicing defect seen in vitro and in vivo. In HeLa cells, hPrp17 is highly concentrated in the nuclear speckles, as is SC35 and many other splicing factors, thus providing further support that this protein also functions as a splicing factor in humans.