In mammalian intron splicing, the mechanism by which the 3' splice site AG is accurately and efficiently identified has remained unresolved. We have previously proposed that the 3' splice site in mammalian introns is located by a scanning mechanism for the first AG downstream of the branch point-polypyrimidine tract. We now present experiments that lend further support to this model while identifying conditions under which competition can occur between adjacent AGs. The data show that the 3' splice site is identified as the first AG downstream from the branch point by a mechanism that has all the characteristics expected of a 5'-to-3' scanning process that starts from the branch point rather than the pyrimidine tract. Failure to recognize the proximal AG may arise, however, from extreme proximity to the branch point or sequestration within a hairpin. Once an AG has been encountered, the spliceosome can still see a limited stretch of downstream RNA within which an AG more competitive than the proximal one may be selected. Proximity to the branch point is a major determinant of competition, although steric effects render an AG less competitive in close proximity (approximately 12 nucleotides). In addition, the nucleotide preceding the AG has a striking influence upon competition between closely spaced AGs. The order of competitiveness, CAG congruent to UAG > AAG > GAG, is similar to the nucleotide preference at this position in wild-type 3' splice sites. Thus, 3' splice site selection displays properties of both a scanning process and competition between AGs based on immediate sequence context. This refined scanning model, incorporating elements of competition, is the simplest interpretation that is consistent with all of the available data.