A 57-kDa readthrough portion of the Tobacco rattle virus (TRV) replicase gene was isolated in two replicates and sequenced. Sequence comparisons showed the isolated sequences to have a homology of 93–94% compared to other TRV isolates at the DNA level and code for proteins with 98–99% similarity to other corresponding TRV sequences at the amino acid level. The isolated sequences were cloned into binary vector constructs, which were transformed to the potato cultivar Matilda. Ten obtained transgenic potato lines were challenged with TRV mediated by the natural nematode vector in greenhouse trials. In a first trial all transgenic lines demonstrated fewer symptoms compared to an untransformed Matilda control. However, the infection pressure in this first trial was in general very low and to get more reliable results a second greenhouse trial with the two most promising lines was performed the following season. In this trial both transgenic lines had significantly fewer symptoms compared to the untransformed control. The symptom reduction for the best line was 78% compared to the control. Thus, introduction into potato of a gene encoding the 57-kDa TRV replicase portion is a possible way to improve resistance to TRV in potatoes.