Drug resistance is a major problem for anti-tuberculous chemotherapy and is one of the main reasons why compounds such as fluoroquinolones are not first line agents. Two recent advances, using a variety of bacteria, encourage refinements of fluoroquinolones. First, resistance arises stepwise from mutations in two independent targets, DNA gyrase and DNA topoisomerase IV. Thus a first step resistant gyrase mutant can be used to screen new fluoroquinolones for derivatives that require two mutations to confer resistance. Since double mutations occur less frequently that single mutations by many orders of magnitude, resistance to the new compounds should arise very rarely. Second, the double mutant concept was experimentally validated: a C8-methoxyl group reduced the acquisition of resistance by at least three orders of magnitude. Two approaches are proposed to define how these principles apply to fluoroquinolone resistance in Mycobacterium tuberculosis. In one, fluoroquinolone structural variants will be examined for their ability to prevent resistance. Prevention of resistance will be correlated with avid killing of moderately resistant strains 1) in pure culture under growing and non-growing conditions, and 2) inside cultured human macrophages. The generality of the conclusions will be tested using a wide variety of clinical isolates of M. tuberculosis. In the other approach, resistance will be studied by examining nucleotide sequences of genes encoding gyrase and topoisomerase IV. Mutants will be selected for resistance to various fluoroquinolone congeners, some of which are already known to elicit different alleles. Selected mutants will be tested for 1)cross-resistance, 2) adaptation to changing environment, and 3) invasion of human macrophages. Already available are many new compounds, two novel assays (killing resistant mutants and prevention of resistance), a human macrophage infection system, and a large collection of clinical isolates of M. tuberculosis. Significant progress is expected toward finding fluoroquinolones that will prevent resistance from arising in M. tuberculosis.