Rokitamycin is the latest semi-synthetic 16-membered ring macrolide introduced into clinical practice. It is characterized by greater hydrophobicity, better bacterial uptake and a slower release, more cohesive ribosomal binding, and a longer post-antibiotic-effect (PAE) than can be observed with other available 14-, 15- and 16-membered ring macrolides. Rokitamycin exerts its activity on strains that harbor inducible erm genes or the efflux gene, mef(A). It has also been reported to be more active in vitro than other 16-membered ring macrolides. However, these recognized features are not fully exploited yet because current automated test procedures used in many microbiological laboratories determine susceptibility only to erythromycin or clarithromycin. Resistance to 16-membered ring macrolides cannot be predicted solely on the basis of known resistance to erythromycin or clarithromycin as revealed by an automated susceptibility assay. At least equally important is the knowledge of the bacterial resistance phenotype. This is underlined by the existence of Gram-positive coccal strains resistant to erythromycin and other 14-,15-membered ring macrolides but susceptible to 16-membered ring macrolides. Since the local prevalence of erythromycin phenotypes is generally unknown but might determine the outcome of treatment, the procedure for identifying the phenotypes in erythromycin-resistant strains (which can be easily and cheaply performed using the two- or three-disk assay) should become routine, at least in the countries in which 16-membered ring macrolides are used. This approach should help to optimize the use of macrolides, improve our knowledge of the local prevalence of phenotypes resistant to erythromycin, and offer the possibility of treating infections caused by certain types of erythromycin-resistant pathogens.