An efficient negative selection procedure is crucial to the isolation of rare homologous recombinants in gene targeting. Although gene targeting is a common practice in lower eukaryotes and is becoming routine in mammals, its application to plants has not been achieved. In this report, we have evaluated an antisense construct against the neomycin phosphotransferase gene (nptII) as a negative selectable marker. The anti-nptII gene construct was able to suppress nptII expression both transiently and in transformed tobacco (Nicotiana tabacum) calli. A construct was made which includes both a hygromycin-resistance gene and the sense plus antisense genes for neomycin phosphotransferase. Hygromy-cin-resistant calli were obtained after Agrobacterium-mediated transformation. Subsequently, hygromycin-resistant calli were tested for kanamycin sensitivity. The growth on kanamycin medium of calli harboring both the sense and antisense gene constructs was retarded, whereas that of control calli transformed with only the sense nptII gene was not inhibited. Southern blot analysis confirmed the presence of both nptII and anti-nptII genes. Northern blot analyses revealed that antisense transcripts of the nptII gene were made and that the level of sense transcripts was greatly reduced in transgenic calli. These results suggest that the anti-nptII gene could potentially be used as a negative selectable marker for gene targeting in plants.