A skin biopsy from a patient with erythema migrans was inoculated into C3H/He mice and into culture medium. A Borrelia garinii strain named Rio1 was isolated from both a direct BSK medium culture and a mouse ear-punch biopsy culture. Inoculating human tissue into mice produced a disease resulting in severe inflammation of the left tibio-tarsal joint, development of perivascular infiltrates as seen in ear-punch biopsies and the spread of spirochaetes along the skin, far from the inoculation site. The isolation of this strain confirms the circulation of this Borrelia species in Spain as a human pathogen, as well as its arthrogenicity in an animal model. The method used to recover strain Rio1 from human tissue is described as rapid and sensitive compared to direct inoculation of tissue into BSK medium.