Ixodes spp. ticks are known to occasionally harbour spiroplasmas – helical mycoplasmas in the class Mollicutes; a previous study in Slovakia reported an overall prevalence of Spiroplasma ixodetis of 3% in Ixodes ricinus. In the present study, extracts of unfed adult I. ricinus ticks collected from vegetation in south-western Slovakia were added to a panel of cell lines derived from I. ricinus and Ixodes scapularis embryos. The cultures were monitored by preparation and examination of Giemsa-stained cytocentrifuge smears at intervals over the subsequent 16–18 months. Spiroplasma-like microorganisms were detected in cultures of both tick species after 2–3 months and subcultured onto fresh, uninfected cells of the appropriate cell line up to seven times. Molecular analysis using PCR assays targeting fragments of the 16S rRNA, ITS and rpoB genes confirmed the identity of the microorganisms as a Spiroplasma sp., with between 98.9% and 99.5% similarity to S. ixodetis. The sequences of the spiroplasmas isolated from three different pools of ticks collected on two different occasions were identical for all three genes tested.