Forty-seven compounds and tetracycline (Tc) structural analogues were tested for their ability to interfere with [3H]Tc uptake in everted inner membrane vesicles derived from Tc-resistant Escherichia coli D1-209, bearing the class B tetracycline resistance efflux protein (Tet protein). For effective inhibition of Tc uptake, the molecule had to have an intact ABCD tetracyclic carbon skeleton and a conjugated phenolic beta-diketone substructure at positions 10-12a with the subsequent development of keto-enol tautomerization. Molecular variations at carbon positions 2, 4, 5, 6, 7, 8, and 9 did not decrease, and some increased, the inhibitory activity as compared to that of Tc. Among these compounds, the highest inhibition of uptake occurred with certain position 6 and 13 derivatives of 5-hydroxytetracycline. In a group of 13-(propylthio) derivatives of 5-OH-Tc [13-propyl, 13-(3-chloropropyl), and 13-(2-carboxyethyl)] there was a correlation between uptake inhibitory activity and antibacterial activity. The 13-(3-chloropropyl) derivative, with the best efflux inhibitory activity, exhibited synergistic activity when tested in combination with doxycycline against Tc-resistant E. coli bearing the class A or B determinant, against Staphylococcus aureus bearing class K, and against Enterococcus faecalis bearing the class L determinant. The 13-propyl analogue also showed high transport blocking activity and showed synergistic antibacterial activity against E. coli bearing the class A determinant and additive activity against the other Tc-resistant bacteria. The synergistic antibacterial activity of these compounds was not shown by the 13-[(2-carboxyethyl)thio] homologue, whose efflux blocking activity was 70-fold less. These findings suggest that multiple sites on the Tc molecule are available for synthetic modification toward the development of an effective Tc blocking agent. Such compounds, used alone or in combination with a standard tetracycline, show improved antibacterial activity.