Five isolates of tomato ringspot virus collected in Virginia were indistinguishable serologically and symptomologically but could be differentiated into three strains by nucleoprotein and nucleic acid analysis. A grape isolate from one location (GSGF), two grape isolates from another location (GBNR, G4NR), a seed-borne soybean isolate (S1B), and a dandelion isolate (D4WR) from the second location were compared. The three grape isolates could be categorized as a single strain even though collected from two different locations. They had middle (M) and bottom (B) nucleoproteins with identical sedimentation coefficients of 121S and 133S, respectively, and buoyant density. The grape isolates also had RNA-1 and RNA-2 of identical Mr (1.97 and 1.82 x 10⁶, respectively) in nondenaturing polyacrylamide gels. The B nucleoprotein of S1B was identical to B of the grape isolates in sedimentation and buoyant density. However, M of S18 had a lower sedimentation coefficient (115S) and buoyant density than M of the grape isolate. The Mrs of HNA-1 and HNA-2 of S1B were 1.97 and 1.87 x10⁶, respectively. Electrophoretic analyses of both S1B and grape RNAs did not substantiate the nucleoprotein data. The unexpected discrepancy between nucleic acid size predicted by biophysical properties and that estimated might be explained by encapsidation of satellite-like RNAs (observed in denaturing gels), or by the unusual secondary structure of the genomic RNAs. No differences in protein were observed since the nucleic acid devoid top component (T) all the isolates WBS identical in sedimentation and density. Isolate D4WR differed from the other isolates by having a single nucleoprotein of 1315 with properties similar to H of the other isolates. Nucleic acid number and size for D4WR were not determined because of contamination with a second virus. Antiserum prepared against pure T of D4WR was positive for all TmR5V strains in microprecipitin but not in agar gel diffusion tests.