The branchpoint sequence and associated polypyrimidine tract are firmly established splicing signals in vertebrates. In plants, however, these signals have not been characterized in detail. The potato invertase mini-exon 2 (9 nt) requires a branchpoint sequence positioned around 50 nt upstream of the 5' splice site of the neighboring intron and a U11 element found adjacent to the branchpoint in the upstream intron (Simpson et al., RNA, 2000, 6:422-433). Utilizing the sensitivity of this plant splicing system, these elements have been characterized by systematic mutation and analysis of the effect on inclusion of the mini-exon. Mutation of the branchpoint sequence in all possible positions demonstrated that branchpoints matching the consensus, CURAY, were most efficient at supporting splicing. Branchpoint sequences that differed from this consensus were still able to permit mini-exon inclusion but at greatly reduced levels. Mutation of the downstream U11 element suggested that it functioned as a polypyrimidine tract rather than a UA-rich element, common to plant introns. The minimum sequence requirement of the polypyrimidine tract for efficient splicing was two closely positioned groups of uridines 3-4 nt long (<6 nt apart) that, within the context of the mini-exon system, required being close (<14 nt) to the branchpoint sequence. The functional characterization of the branchpoint sequence and polypyrimidine tract defines these sequences in plants for the first time, and firmly establishes polypyrimidine tracts as important signals in splicing of at least some plant introns.