In the screening program for microbial transformation of 16-membered macrolide antibiotics, Streptomyces thermotolerans ATCC 11416 was found to acylate the hydroxyl group at the 3- and 4"-positions of 16-membered macrolide antibiotics including tylosin. This paper reports eight acylated derivatives of tylosin: 4"-butyryltylosin, 4"-isovaleryltylosin, 3-acetyltylosin, 3-acetyl-4"-butyryltylosin, 3-acetyl-4"-isovaleryltylosin, 3-propionyltylosin, 3-propionyl-4"-butyryltylosin and 3-propionyl-4"-isovaleryltylosin. The antibacterial spectra of these new tylosin derivatives were determined by the agar dilution method (compared with tylosin, erythromycin, leucomycin A3, etc.), focusing on the role of the 4"-acyl group in antibacterial activity against macrolide-resistant strains. The results showed that the eight acylated derivatives inhibited the growth of macrolide-sensitive Gram-positive bacteria similarly to other macrolide antibiotics but not Gram-negative bacteria. For macrolide-resistant strains of Staphylococcus and Streptococcus, the new acylated tylosin derivatives displayed increased activity; specifically, 4"-acylation enhanced antibacterial activity to a greater extent than 3-acylation (e.g., 4"-isovaleryltylosin showed stronger activity than 3-acetyltylosin and tylosin). Thus, the 4"-acyl group plays a significant role in the antibacterial activity of these tylosin derivatives against macrolide-resistant bacteria. In contrast, the 4"-acyl group of leucomycins has no important effect on activity against resistant bacteria, suggesting tylosin itself has latent specific activity against macrolide-resistant bacteria that is expressed by 4"-acylation.