The antimicrobial peptide MSI-99, an analog of magainin 2, was expressed via the chloroplast genome to obtain high levels of expression in transgenic tobacco (Nicotiana tabacum var. Petit Havana) plants. Polymerase chain reaction products and Southern blots confirmed integration of MSI-99 into the chloroplast genome and achievement of homoplasmy, whereas northern blots confirmed transcription. Contrary to previous predictions, accumulation of MSI-99 in transgenic chloroplasts did not affect normal growth and development of the transgenic plants. This may be due to differences in the lipid composition of plastid membranes compared with the membranes of susceptible target microbes. In vitro assays with protein extracts from T1 and T2 plants confirmed that MSI-99 was expressed at high levels to provide 88% (T1) and 96% (T2) inhibition of growth against Pseudomonas syringae pv tabaci, a major plant pathogen. When germinated in the absence of spectinomycin selection, leaf extracts from T2 generation plants showed 96% inhibition of growth against P. syringae pv tabaci. In addition, leaf extracts from transgenic plants (T1) inhibited the growth of pregerminated spores of three fungal species, Aspergillus flavus, Fusarium moniliforme, and Verticillium dahliae, by more than 95% compared with non-transformed control plant extracts. In planta assays with the bacterial pathogen P. syringae pv tabaci resulted in areas of necrosis around the point of inoculation in control leaves, whereas transformed leaves showed no signs of necrosis, demonstrating high-dose release of the peptide at the site of infection by chloroplast lysis. In planta assays with the fungal pathogen, Colletotrichum destructivum, showed necrotic anthracnose lesions in non-transformed control leaves, whereas transformed leaves showed no lesions. Genetically engineering crop plants for disease resistance via the chloroplast genome instead of the nuclear genome is desirable to achieve high levels of expression and to prevent pollen-mediated escape of transgenes.