Many protein-protein interactions involved in cell signalling, cell adhesion and regulation of transcription are mediated by short alpha-helical recognition motifs with the sequence Leu-Xaa-Xaa-Leu-Leu (LXXLL, where Xaa is any amino acid). Originally observed in cofactors that interact with hormone-activated nuclear receptors, LXXLL motifs are now known to occur in many transcription factors, including the STAT family, which transmit signals from activated cytokine receptors at the cell surface to target genes in the nucleus. STAT 6 becomes activated in response to IL-4 and IL-13, which regulate immune and anti-inflammatory responses. Structural studies have revealed how an LXXLL motif located in 2.5 turns of an alpha-helical peptide derived from STAT 6 provide contacts through the leucine side chains to the coactivator of transcription, NCoA-1. However, since many protein-protein interactions are mediated by LXXLL motifs, it is important to understand how specificity is achieved in this and other signalling pathways. Here, we show that energetically important contacts between STAT 6 and NCoA-1 are made in residues that flank the LXXLL motif, including the underlined residues in the sequence LLPPTEQDLTKLL. We also demonstrate how the affinity for NCoA-1 of peptides derived from this region of STAT 6 can be significantly improved by optimising knobs-into-holes contacts on the surface of the protein. The results provide important new insights into the origins of binding specificity, and might be of practical value in the design of novel small-molecule inhibitors of this important protein-protein interaction.