The activity of rokitamycin, a new macrolide with a 16-member ring, was tested against Borrelia burgdorferi in vitro. The antibiotic had a lower MIC at which 50% of the isolates are inhibited than erythromycin, the parent 14-member macrolide, but the same MIC at which 50% of the isolates are inhibited as the other recent 14- and 15-member macrolides, like clarithromycin and azithromycin. The MBC was equal to the MIC at which 50% of the isolates are inhibited, so rokitamycin can be considered bactericidal against B. burgdorferi. The sensitivity of the Borrelia strains tested was not correlated with the particular species Burgdorferi sensu stricto, B. garinii, and B. afzelii or with the number of subcultures of the isolates.