ABSTRACT Previously we cloned two human RCC1 cDNAs that differed in their noncoding region. In this study, we have found new human and hamster RCC1 cDNAs, which have an even more different coding region from that of the previously cloned RCC1 cDNAs yet can complement the RCC1 mutation in the tsBN2 cell line. The newly found RCC1 cDNAs encode a protein (designated as RCC1-I) that has an insertion of 31 (human) and 13 (hamster) amino acids at valine25 in the N-terminal region outside the RCC1-seven repeat. The inserted nucleotide sequence was searched for, within the human RCC1 genomic sequence that had already been determined, and was found to be located between the 6th and 7th exons, designated as the 6′ exon. Both the 5′ and 3′ ends of the 6′ exon correspond to the GT-AG rules for splicing, indicating that human RCC1-I mRNAs are produced by alternative splicing. The finding that both humans and hamsters have the insertion at the same RCC1 site suggests that the pattern of alternative splicing in the RCC1 gene has been conserved through evolution.