Many proteins are anchored to lipid bilayer membranes through a combination of hydrophobic and electrostatic interactions. In the case of the membrane-bound nonreceptor tyrosine kinase Src from Rous sarcoma virus, these interactions are mediated by an N-terminal myristoyl chain and an adjacent cluster of six basic amino-acid residues, respectively. In contrast with the acyl modifications of other lipid-anchored proteins, the myristoyl chain of Src does not match the host lipid bilayer in terms of chain conformation and dynamics, which is attributed to a tradeoff between hydrophobic burial of the myristoyl chain and repulsion of the peptidic moiety from the phospholipid headgroup region. Here, we combine thermodynamic information obtained from isothermal titration calorimetry with structural data derived from (2)H, (13)C, and (31)P solid-state nuclear magnetic resonance spectroscopy to decipher the hydrophobic and electrostatic contributions governing the interactions of a myristoylated Src peptide with zwitterionic and anionic membranes made from lauroyl (C12:0) or myristoyl (C14:0) lipids. Although the latter are expected to enable better hydrophobic matching, the Src peptide partitions more avidly into the shorter-chain lipid analog because this does not require the myristoyl chain to stretch extensively to avoid unfavorable peptide/headgroup interactions. Moreover, we find that Coulombic and intrinsic contributions to membrane binding are not additive, because the presence of anionic lipids enhances membrane binding more strongly than would be expected on the basis of simple Coulombic attraction., (Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.)