Replication Protein A (RPA) is a single stranded DNA-binding protein involved in DNA metabolic activities such as replication, repair, and recombination. RPA-Interacting Protein alpha (RIPalpha) was originally identified as a nuclear transporter of RPA in Xenopus. The human RIPalpha gene encodes several splice isoforms, of which hRIPalpha and hRIPbeta are the major translation products in vivo. However, limited information is available about the alternative splicing of RIPalpha in eukaryotes, apart from that in humans. In this study, we examined the alternative splicing of RIPalpha in the Drosophila, Xenopus, and mouse system. We showed that the number of splice isoforms of RIPalpha was species-specific, and displayed a tendency to increase in higher eukaryotes. Moreover, a mouse ortholog of hRIPbeta, mRIPbeta2, was not SUMOylated, in contrast to hRIPbeta. Based on these results, we suggest that the RIPalpha gene gains more splice isoforms and additional modifications after molecular evolution. [BMB reports 2009; 42(1): 22-27].