Leaves of symptomless Fragaria ananassa Duch cv. Cacanská raná were grafted onto Fragaria vesca indicator clones. Thirty-five of 72 grafted indicator plants developed leaf mottle symptoms. Isometric virus-like particles were observed in purified preparations from symptomatic leaves of F. vesca. The latter were mechanically inoculated to herbaceous host plants. A virus was successfully purified from Nicotiana occidentalis 37 B symptomatic plants by differential and sucrose density gradient centrifugations and a polyclonal antiserum to the virus was prepared. On the basis of serological reactions, symptomatology on herbaceous hosts and electron microscopy studies the virus was identified as tobacco necrosis virus (TNV) D-strain. This is the first isolation of TNV from strawberry leaves and its first finding on strawberry in the Czech Republic. The new experimental hosts N. aucalis, N. bentamiana, N. occidentalis 37 B (systemic hosts), and Ammobium alatum, N. bigelovi, Petunia hybrida (local hosts) for TNV are reported. These results may not exclude the presence of strawberry mottle virus as a causal agent of mottle symptoms in the tested plant samples. Further research is necessary to clarify the aetiology of the strawberry mottle.