The yeast poly(A)-binding protein (Pab1p) contains four RNA recognition motifs (RRMs). Site-directed mutations were introduced into each of these RRMs in order to investigate their relative contributions to specific and non-specific RNA binding, and to determine the consequences of these mutations on the ability of Pab1p to support viability. Specifically, a charged and an aromatic residue that were predicted to be involved in RNA binding were mutated in each RRM. These mutations revealed that the second RRM is primarily responsible for poly(A) binding, while the fourth RRM is primarily responsible for non-specific polypyrimidine RNA binding. The mutated aromatic residues in each RRM contributed to both modes of binding whereas the mutated charged residues contributed primarily to non-specific RNA binding. RNA binding in vivo correlated with the in vitro binding measurements. Furthermore, RNA binding, but not high-affinity poly(A) binding, correlated with the ability of Pab1p to sustain yeast cell viability. These data suggest that a single aromatic substitution in Pab1p can significantly reduce its RNA binding ability, that the capacity of Pab1p to bind poly(A) as well as other RNAs is mediated by distinct residues within different RRMs, and that Pab1p does not require high affinity poly(A) tail binding to perform its essential function.