Since the discovery of RNA recombination in polioviruses, there has been a general belief that this mechanism operates only in positive-sense RNA viruses. Recently, studying wild-type Tula hantavirus, we observed a mosaic-like structure of the S RNA segment that was consistent with generation by recombination between viruses from two genetic lineages. Here we show transfection-mediated rescue of Tula virus carrying recombinant S RNA segment. Independent attempts yielded S RNA molecules of similar structure; the majority of them carried a break point located close to one of the break points suggested for natural recombinants. Recombinant virus purified from the original variant was able to grow to the same titers in cell culture and showed the same characteristic immunofluorescence pattern when stained for the nucleocapsid protein. While competent, the recombinant virus appeared to be slightly less competitive than the wild type. Sequence analysis of the S cDNA clones obtained from the purified recombinant virus confirmed that all S RNA molecules were of recombinant origin. This provides the first example of a negative-sense RNA virus constructed using homologous recombination.