Ketolides represent the latest generation of macrolide antibiotics, displaying improved activities against some erythromycin resistant strains, while maintaining their activity against erythromycin susceptible ones. In this study we present a new ketolide K-1325 that carries an alkyl-aryl side chain at C-13 of the lactone ring. According to our genetic and biochemical studies K-1325 binds within the nascent polypeptide exit tunnel, at a site previously described as the primary attachment site of all macrolide antibiotics. Compared with telithromycin, K-1325 displays enhanced antimicrobial activity against wild type E.coli strains, as well as against strains bearing the U2609C mutation in 23S rRNA. Chemical protection experiments showed that the aryl-alkyl side chain of K-1325 interacts specifically with helix 35 of 23S rRNA, a fact leading to an increase affinity of U2609C mutant ribosomes for the drug, and rationalizing the enhanced effectiveness of this new ketolide.