The susceptibilities of 228 penicillin- and erythromycin-susceptible and -resistant pneumococci to RU 64004, a new ketolide, were tested by agar dilution, and the results were compared with those for penicillin G, erythromycin, azithromycin, clarithromycin, rokitamycin, clindamycin, pristinamycin, ciprofloxacin, sparfloxacin, trimethoprim-sulfamethoxazole, doxycycline, chloramphenicol, cefuroxime, ceftriaxone, imipenem, and vancomycin. RU 64004 was very active against all strains tested, with MICs at which 90% of the isolates are inhibited (MIC90s) of 0.016 microg/ml for erythromycin-susceptible strains (MIC, < or = 0.25 microg/ml) and 0.25 microg/ml for erythromycin-resistant strains (MIC, > or = 0.5 microg/ml). All other macrolides had MIC90s of 0.03 to 0.25 and > or = 128 microg/ml for erythromycin-susceptible and -resistant strains, respectively. Among erythromycin-resistant strains, clindamycin MICs for 28 of 91 (30.7%) were < or = 0.125 microg/ml. Pristinamycin MICs for all strains were < or = 1.0 microg/ml. MIC90s of ciprofloxacin and sparfloxacin were 4.0 and 0.25 microg/ml, respectively, and were unaffected by susceptibility to penicillin or erythromycin. Vancomycin and imipenem inhibited all strains at < or = 0.5 and < or = 0.25 microg/ml, respectively. MICs of cefuroxime and cefotaxime rose with those of penicillin G. MICs of trimethoprim-sulfamethoxazole, doxycycline, and chloramphenicol were variable but were generally higher for penicillin- and erythromycin-resistant strains. RU 64004 is the first member of the macrolide group which has low MICs for erythromycin-resistant pneumococci.