The activity of rokitamycin, a 16-membered macrolide, was compared with other macrolides and agents used to treat respiratory infections. Rokitamycin had in vitro activity against streptococci and Streptococcus pneumoniae comparable to the other macrolides, inhibiting most organisms at less than 0.03 to 0.5 microgram/ml. It was the most active macrolide agent against Staphylococcus aureus, inhibiting 90% at 1 microgram/ml. Macrolide-resistant streptococci and staphylococci in which resistance was inducible were inhibited, but constitutively resistant Gram-positive bacteria were resistant. Rokitamycin was less active than erythromycin against Haemophilus influenzae, but had activity comparable to erythromycin against Moraxella catarrhalis and Neisseria gonorrhoeae. It inhibited Clostridium spp. and peptostreptococci, but had poor activity against Bacteroides species. Rokitamycin was bactericidal for streptococci and staphylococci, but not for enterococci. Overall rokitamycin has in vitro activity comparable to currently available 14-membered macrolides. Its clinical utility will be influenced by the degree of metabolism to less active metabolites since 70% is rapidly metabolized.