The objective of this study is to determine the bioavailability of clay-sorbed tetracycline to bacteria for activation of antibiotic resistance genes. Tetracyclines are a class of antimicrobials used extensively in livestock production. A large portion of tetracyclines administered to animals are excreted and subsequently released into the environment, which are strongly associated with soil minerals. This raises concerns whether tetracyclines bound to soils are bioavailable to bacteria and activate their antibiotic resistance genes. In this study, we used E.coli whole-cell bioreporter as a target bacterium to determine the bioavailability of smectite clay-sorbed tetracycline under varying conditions. Tetracycline sorbed by the clay was desorbed to aqueous phase, and became bioavailable to the bacterium, which is the primary pathway of bacterial uptake of tetracycline. However, the tetracycline strongly associated with clay surfaces is still bioavailable to the bacterium as indicated by triggering the antibiotic resistance genes.