We present a detailed comparative analysis of the PDZ domains of the human LAP proteins Erbin, Densin-180, and Scribble and the MAGUK ZO-1. Phage-displayed peptide libraries and in vitro affinity assays were used to define ligand binding profiles for each domain. The analysis reveals the importance of interactions with all four C-terminal residues of the ligand, which constitute a core recognition motif, and also the role of interactions with more upstream ligand residues that support and modulate the core binding interaction. In particular, the results highlight the importance of site(-1), which interacts with the penultimate residue of ligand C termini. Site(-1) was found to be monospecific in the Erbin PDZ domain (accepts tryptophan only), bispecific in the first PDZ domain of ZO-1 (accepts tryptophan or tyrosine), and promiscuous in the Scribble PDZ domains. Furthermore, it appears that the level of promiscuity within site(-1) greatly influences the range of potential biological partners and functions that can be associated with each protein. These findings show that subtle changes in binding specificity can significantly alter the range of biological partners for PDZ domains, and the insights enhance our understanding of this diverse family of peptide-binding modules.