The mechanism of action of the substance B44P, an antibacterial antibiotic, was studied with growing cells of E. coli B and St. aureus 209P. This antibiotic was found to inhibit protein and RNA synthesis in these cells. With a cell-free system using S-30 fraction of E. coli B, this antibiotic was shown not to affect protein synthesis, in absence of RNA synthesis. Consequently, it was concluded that the substance B44P primarily inhibits RNA synthesis in bacterial cells. The difference in the mechanism of RNA synthesis inhibition by the substance B44P and actinomycin D was discussed.