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Retroviruses utilize balanced splicing to express multiple proteins from a single primary transcript. A number of cis -acting signals help maintain this balance, including the branch point sequence (BPS), polypyrimidine tract (PPyT) and sequences within the downstream exon. In general, regulated splicing requires weak splicing signals and we have previously shown the same requirement for the simple retrovirus, avian sarcoma virus (ASV). Here we take advantage of the requirement for balanced splicing in retroviral replication to examine the sequence constraints of an intronic splicing element. Selection for replication competence makes it possible to amplify and identify functional sequences from a pool of all possible sequences. In this report we examine the role of pyrimidines within the PPyT. Our results provide in vivo confirmation that the functional strength of a PPyT is related to its length and uridine content and that the PPyT plays a role in the second step of the splicing reaction. We also show that the minimal distance between the 3'-splice site and the BPS in this system is 16 nt. With modification, the selection system described here can be used to examine the sequence constraints of other exonic or intronic splicing elements in vivo .