Isolates of Vibrio harveyi, a prawn pathogen, have demonstrated multiple antibiotic resistance to commonly used antimicrobial agents, such as oxytetracycline. In this paper, we describe the cloning and characterization of two tetracycline resistance determinants from V. harveyi strain M3.4L. The first resistance determinant, cloned as a 4,590-bp fragment, was identical to tetA and flanking sequences encoded on transposon Tn10 from Shigella flexneri. The second determinant, cloned as a 3,358-bp fragment in pATJ1, contains two open reading frames, designated tet35 and txr. tet35 encodes a 369-amino-acid protein that was predicted to have nine transmembrane regions. It is a novel protein which has no homology to any other drug resistance protein but has low levels of homology (28%) to Na(+)/H(+) antiporters. Transposon mutagenesis showed that tet35 and txr were required for tetracycline resistance in a heterologous Escherichia coli host. Tetracycline accumulation studies indicate that E. coli carrying tet35 and txr can function as an energy-dependent tetracycline efflux pump but is less efficient than TetA.