9, 3"-Di-O-acetyl midecamycin (Mom) showed in vitro antibacterial activity against clinical isolates of Staphylococcus aureus and was effective against some of the strains resistant to erythromycin (Em) and josamycin (Jm). The distribution pattern of the level of drug-sensitivity of the isolates to Mom was similar to that of Jm but different from that of Em. Mom- or Jm-resistant strains gradually or rapidly developed among S. aureus strains, that were sensitive or resistant to other macrolide antibiotics. There was no significant difference between Mom and Jm as regards the rate of development of resistant strains. These mutants were always resistant to both Mom and Jm. Mom, like Jm, was effective against Em-inducible strains of S. aureus. The in vivo study demonstrated that Mom was more potent than Jm, similar in its potency to Em against systemic staphylococcal infections, and that it was effective against the infection due to an Em-resistant clinical isolate of S. aureus. Mom was more effective than either Jm or Em against a staphylococcal kidney infection in mice.