The accumulation of macrolide antibiotics in Bacteroides fragilis ATCC 25285 was increased in the order erythromycin, josamycin, and rokitamycin, depending on hydrophobicity. The half-times of efflux were also prolonged in the same order. Furthermore, MICs of the antibiotics were correlated with the extent of hydrophobicity. These findings suggest that the macrolide antibiotics are accumulated in B. fragilis by means of their hydrophobic properties, and the efficient accumulation of the drugs may explain the susceptibility of this gram-negative bacterium to macrolides.