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Spiroplasmas are vertically transmitted endosymbionts of arthropods, including ticks, and pathogens of some arthropods and plants. They are helical mycoplasmas belonging to the class Mollicutes. In ticks, they can be found in the salivary glands, gut, and reproductive organs. Spiroplasma species have been detected in, and/or isolated in vitro from, ticks in Germany, Slovakia, Norway and several other European countries. However, to our knowledge, there are no reports suggesting their presence in Swedish ticks. This study aimed to detect and isolate Spiroplasma spp. from Swedish Ixodes ricinus ticks using tick cell lines as a substrate. Tissues from adult female ticks (n=28) collected from dogs and cats in different regions of Sweden were inoculated into tick cell lines derived from Rhipicephalus microplus (BME/CTVM23) and I. ricinus (IRE/CTVM19). A conventional cytocentrifugation technique combined with Giemsa staining was used to visualize the presence of bacteria. PCR analysis was also performed, using Spiroplasma genus-specific primers targeting the 16S rRNA and rpoB genes. The results suggest that 61.9% of BME/CTVM23 cell cultures are positive for Spiroplasma spp. in both Giemsa-stained cytocentrifuge smears and PCR (16S rRNA). Spiroplasma spp. were detected intracellularly in vacuoles of BME/CTVM23 cells as well as by 16S rRNA PCR 76 days after inoculation with I. ricinus internal organs. Isolation of Spiroplasma spp. will help us to study more closely the bacteria and their influence on the tick's fitness and reproduction, and their role in the persistence of pathogenic microorganisms that are hosted by ticks.