The 5S ribosomal RNA (rRNA) genes represent one of the most abundant gene families in eukaryotic genomes and have been a model system for the study of molecular organization and evolution of multigene families in eukaryotes. This paper reports a detailed characterization of the 5S rRNA genes of tomato (Lycopersicon esculentum) with respect to chromosome assignment, chromosomal localization, copy number, and physical size. By restriction fragment length polymorphism, the tandemly repeated 5S rRNA genes were assigned to a region of chromosome 1 of tomato. These results were confirmed by in situ hybridization onto tomato metaphase chromosomes. The single hybridization signal was localized to the short arm of chromosome 1, in a region close to the centromere. Based on reconstruction experiments, it was estimated that the 400-bp repeating unit occurs in approximately 1000 copies per haploid genome. Physical characterization of the entire locus was then performed by means of pulsed-field gel electrophoresis. Digestion of high molecular weight DNA of tomato with restriction enzymes such as PvuII, ClaI, and BglII resulted in a very prominent band with a size between approximately 450 and 600 kb. This value closely matched the estimated size of the gene cluster based on reconstruction experiments. The data therefore suggest that all the 5S rRNA genes in tomato occur in a single, continuous array, uninterrupted by unrelated sequences.Key words: 5S rRNA genes, organization, Lycopersicon esculentum, in situ hybridization, pulsed-field gel electrophoresis.