Widespread antibiotic resistance is a major public health concern, and plants represent an emerging antibiotic exposure route. Recent studies indicate that crop plants fertilized with antibiotic-laden animal manure accumulate antibiotics, however, the molecular mechanisms of antibiotic entry and subcellular partitioning within plant cells remain unknown. Here we report that mutations in the Arabidopsis locus Multiple Antibiotic Resistance (MAR1) confer resistance, while MAR1 overexpression causes hypersensitivity to multiple aminoglycoside antibiotics. Resistance is highly specific for aminoglycosides and does not extend to antibiotics of other classes, including the aminocyclitol, spectinomycin. Yeast expressing MAR1 are hypersensitive to the aminoglycoside, G418, but not to chloramphenicol or cycloheximide. MAR1 encodes a protein with 11 putative transmembrane domains with low similarity to ferroportin1 from Danio rerio. A MAR1:YFP fusion protein localizes to the chloroplast, and chloroplasts from plants overexpressing MAR1 accumulate more of the aminoglycoside, gentamicin, while mar1-1 mutant chloroplasts accumulate less than wild type. MAR1 overexpression lines are slightly chlorotic, and this chlorosis is rescued by application of exogenous iron. MAR1 expression is also downregulated by low iron. Taken together, these data suggest that MAR1 is a plastid transporter that is likely to be involved in cellular iron homeostasis, and allows opportunistic entry of multiple antibiotics into the chloroplast. mar1 mutants represent an interesting example of plant antibiotic resistance that is based on the restriction of antibiotic entry into a subcellular compartment. Knowledge about this process – and other processes of antibiotic entry – could enable the production of crop plants that are incapable of antibiotic accumulation, aid in development of phytoremediation strategies for decontamination of water and soils polluted with antibiotics, and further the development of new plant-based molecular markers. The work described here also contributes to our understanding of how plants interact with the antibiotics they encounter, both in the laboratory (where aminoglycosides such as kanamycin are used heavily to select for transgenics) and in the natural environment. / text