The spliceosomal proteins U1A and U2B′′ each use a homologous RRM domain to bind specifically to their respective snRNA targets, U1hpII and U2hpIV, two stem-loops that are similar yet distinct in sequence. Previous studies have shown that binding of U2B′′ to U2hpIV is facilitated by the ancillary protein U2A′, whereas specific binding of U1A to U1hpII requires no cofactor. Here we report that U2A′ enables U2B′′ to distinguish the loop sequence of U2hpIV from that of U1hpII but plays no role in stem sequence discrimination. Although U2A′ can also promote heterospecific binding of U1A to U2hpIV, a much higher concentration of the ancillary protein is required due to the ∼500-fold greater affinity of U2A′ for U2B′′. Additional experiments have identified a single leucine residue in U1A (Leu-44) that is critical for the intrinsic specificity of this protein for the loop sequence of U1hpII in preference to that of U2hpIV. Our data suggest that most of the difference in RNA-binding specificity between U1A and U2B′′ can be accounted for by this leucine residue and by the contribution of the ancillary protein U2A′ to the specificity of U2B′′.