Transport of tetracycline into Escherichia coli was studied by two methods, one involving an absolute determination of accumulated drug and the other a fluorescence assay. Tetracycline uptake was nonsaturable when assayed by the absolute method, but fluorescence enhancement was maximal at an initial external tetracycline concentration of about 200 microM. The two transport assays also gave different results for the pH optimum of tetracycline transport. The absolute method indicated a pH optimum of 7.0 to 8.0, whereas the fluorescence method gave a value of 5.5 to 6.0. These data indicate that the fluorescence assay is of limited value in certain situations.