Mammalian splicing factor SF3a consists of three subunits of 60, 66, and 120 kDa and functions early during pre-mRNA splicing by converting the U2 snRNP into its active form. A cDNA encoding the 120-kDa subunit of SF3a has been cloned. The SF3a120 gene was localized to human chromosome 22, and three mRNAs of 3.2, 3.8, and 5.7 kb are ubiquitously expressed. The N-terminal half of the deduced SF3a120 amino acid sequence contains a tandemly repeated motif (the SURP module) that has recently been identified in the essential splicing factor PRP21p of Saccharomyces cerevisiae, the Drosophila alternative splicing regulator suppressor-of-white-apricot, and four proteins from nematodes and mammals; the C-terminal half is organized into a proline-rich region and a ubiquitin-like domain. The spacing between the SURP modules and the protein's essential function in constitutive splicing identify SF3a120 as the mammalian homologue of yeast PRP21p. Binding studies with truncated derivatives of SF3a120 revealed that the SURP domains function in binding to SF3a60, whereas a region of 130 amino acids C-terminal to these domains is essential for contacts with SF3a66.