Background selection can affect variation at protein loci by removing closely linked deleterious mutants. Nevertheless, the influence of background selection in shaping patterns of linkage disequilibrium among protein loci along chromosomes is still little known. We report here the first study assessing the effects of background selection on linkage disequilibrium between protein loci along a single chromosome. Specifically, patterns of disequilibrium between pairs of 15 loosely linked protein loci distributed along the third chromosome of Drosophila melanogaster were characterized in a large sample (605) of wild chromosomes from the natural population of Santa Cruz de Rivadulla (Spain), classified as lethal or non-lethal on the basis of egg-to-adult viability in homozygosis. We found that background selection against lethal-bearing chromosomes changes remarkably the distribution and patterns of disequilibrium along the chromosome. In particular, the amount of disequilibrium between functionally related loci is significantly lower in lethal than in non-lethal chromosomes. These observations suggest that background selection against lethal genes erodes epistatic fitness interactions between protein loci, taking into account that long-distance disequilibrium between functionally related loci is indicative of epistasis. Overall, disequilibrium along the third chromosome appears to be influenced by very different selective forces operating indirectly (background selection) and directly (epistatic selection) on protein loci. The present investigations are relevant for understanding the multilocus architecture of the functional genome.