Using a tobacco ubiquitin cDNA clone as a probe, a genomic clone in EMBL3 coding for a tobacco polyubiquitin protein was isolated. Southern blot hybridization of the genomic clone with the cDNA clone identified a BamHI/EcoRI fragment of 2.5 kb to contain the coding region of polyubiquitin, and thus the fragment was subcloned into a plasmid vector. Nucleotide sequence determination of the clone identified an open reading frame for the four head-to-tail repeats of ubiquitin monomer of 76 amino acids interrupted by an intron sequence of 55 nucleotides. The four ubiquitin units were completely conserved except for the extra glutamine at the carboxy terminus of the last ubiquitin monomer. At the 5'-region upstream of the open reading frame, a sequence of 630 nucleotides was determined. In this region, well-known regulatory sequences such as the CCAAT box, TATA box and heat-shock elements could not be located; instead, a region very rich in C and T and repeats of CA was noticed. In the 3'-downstream region of the open reading frame, a sequence of 474 nucleotides was determined which contained putative polyadenylation signals and a GU-rich region.