The organization of the nuclear-encoded 18S, 5.8S, and 25S ribosomal RNA genes (ribosomal DNA; rDNA) of 21 New World species from different sections of the genus Solanum, of two Old World Solanum species, and of representatives of other Solanaceae (Nicotiana, Atropa, Datura, Physalis, and Capsicum) was analyzed by restriction enzyme mapping using different rDNA specific hybridization probes. All Solanum species investigated exhibited rDNA repeats between 8.7 and 9.3 kb in length; the only exception was S. neorossii with a repeat length of 10.3 kb. Sequence heterogeneity was observed mostly in the intergenic spacer (IGS) region. Restriction sites for EcoRI and DraI in the spacer sequences were found to be characteristic for the New World species of the genus Solanum and for Lycopersicon esculentum. An additional XbaI site was detected in the spacer region of two nontuber-bearing species, S. brevidens and S. etuberosum (subsection Estolonifera Hawkes; series Etuberosa), as well as in the primitive tuber-bearing species of the series Pinnatisecta and Polyadenia (subsection Potatoe G. Don), thus demonstrating that these Mexican species are separated from the other tuber-bearing species but are closely linked to the nontuber-bearing Estolonifera group. Two EcoRI sites mapped at the 3' end of the 25S rRNA coding region seem to be characteristic for members of the Solanaceae; the first EcoRI site is apparently methylated in approximately 50% of the rDNA repeats. Southern hybridization with an IGS fragment of Solanum tuberosum as hybridization probe and nucleotide sequence analysis of the phylogenetically informative 3' end of the 25S rDNA support the assumption that the New World species of the genus Solanum are closely related to Lycopersicon (tomato) in contrast with other Solanaceae investigated, Nicotiana, Atropa, Datura, Physalis, and Capsicum.(ABSTRACT TRUNCATED AT 250 WORDS)