Erythromycin is a macrolide antimicrobial chemically comprised of a 14-membered lactone ring substituted with a neutral (cladinose) and an amino (desosamine) sugar. Recently, a number of new macrolide molecules have been identified containing either 14-, 15- or 16-membered substituted lactone rings. In this study the authors have determined the in vitro activity of roxithromycin and clarithromycin (both 14-membered macrolides), azithromycin (a 15-membered macrolide or azalide) and midecamycin acetate (a 16-membered macrolide) against clinical isolates of Staphylococcus spp., (including methicillin-susceptible and -resistant isolates), Legionella spp., Mycoplasma spp. and Ureaplasma urealyticum. Minimum inhibitory concentrations of the macrolides for the clinical isolates of Staphylococcus spp. examined were widely distributed. However, midecamycin acetate retained activity against those isolates of Staphylococcus spp. exhibiting inducible resistance to erythromycin and the other macrolides tested. Isolates characterised by constitutive resistance to erythromycin were also resistant to midecamycin acetate. All of the macrolides were very active against Legionella spp., with clarithromycin demonstrating the greatest potency (MIC range: less than or equal to 0.03-0.06 mg/l). Isolates of Mycoplasma pneumoniae and Ureaplasma urealyticum were susceptible to all of the macrolides tested. However, erythromycin, roxithromycin, clarithromycin and azithromycin were poorly active against isolates of Mycoplasma hominis. By contrast, the same isolates were susceptible (MIC range: 0.008-0.12 mg/l) to midecamycin acetate.