Four root-specific cDNA clones and their corresponding genomic clones have been isolated from tobacco (Nicotiana tabacum) by a novel differential hybridization procedure. The genes are expressed at high levels in roots and are not detectable in leaves. The cDNAs are encoded by small gene families of two to four members. Transcription experiments with isolated nuclei demonstrate that the genes are, at least in part, transcriptionally regulated. Constructions in which 1.4 kilobase pairs of 5' flanking region of one of the root-specific genes was fused to a reporter gene (beta-glucuronidase) were transformed into tobacco. beta-Glucuronidase activity in transgenic plants was localized in the roots, demonstrating the cis-acting sequences regulating root-specific expression are present on the 5' flanking region.