DNA was isolated from crude nuclear pellets prepared from Rutgers tomato plant leaf tissue from both potato spindle tuber viroid (PSTV)-infected and uninfected plants. Following digestion by the DNA restriction enzymes EcoRI, XbaI, or BamHI, the DNA was fractionated by agarose gel electrophoresis, transferred to nitrocellulose filters, and incubated with a variety of 125I-labeled RNA probes under conditions which permit hybridization. Analysis of autoradiogram patterns and fingerprints of the RNA recovered from individual bands following hybridization of the three tomato cytoplasmic ribosomal species, 5, 18, and 26 S, permitted us to construct a consistent restriction map about 8800 base pairs in length containing the 18 and 26 S species and to demonstrate that the 5 S gene clusters are not linked to the 18 and 26 S regions and themselves have a repeat length of about 350 base pairs. These studies formed the background for a search for hybridization between 125I-labeled PSTV and DNA from uninfected and PSTV-infected tomato plants. Significant hybridization was observed when conventionally prepared viroid was used as the probe in Southern hybridization experiments. However, fingerprint analysis of the RNA recovered from these bands demonstrated that this hybridization was due to the presence of ribosomal RNA species in the viroid preparation. When the viroid was subjected to further purification steps (electrophoresis in gels containing 8 M urea) prior to hybridization, all traces of hybridization due to host contaminants disappeared and no viroid-specific bands could be detected, even upon prolonged exposure of the autoradiograms. Under these same hybridization conditions, we could detect as little as one-quarter copy of a gene 550 base pairs in length.