Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] is one of the world's most valuable timber species and is widely used in reforestation. Therefore there is considerable interest in understanding its genetic structure. In conifers approximately 75% of the genome is composed of repetitive DNA. Thus for conifers characterization of repetitive DNA is a significant part of genome analysis. We have characterized the organization of 5S rRNA genes in Douglas-fir at both the molecular and chromosome levels. 5S DNA repeat units containing the coding sequence for 5S rRNA and the nontranscribed spacer (NTS) were cloned using PCR. Sequencing and Southern hybridization revealed repeat units of 888 and 871 bp in length, the latter with a 17 bp deletion in the NTS. The coding region showed high homology with other eukaryotic 5S rRNA genes. A 35 bp region of the NTS immediately upstream of the 5' end of the coding region showed high similarity to other conifers but not to other published plant 5S rDNA sequences. Physical mapping of 5S rDNA by fluorescent in situ hybridization using a biotinilated homologous probe revealed a single subtelomeric site on one pair of large metacentric chromosomes.