Theileriosis is a tick‐borne disease caused by intracellular protozoa of the genus Theileria. The most important species in cattle are Theileria annulata and Theileria parva. Both species transform leucocyte host cells, resulting in their uncontrolled proliferation and immortalization. Vaccination with attenuated T. annulata‐infected cell lines is currently the only practical means of inducing immunity in cattle. Culture media for Theileria spp. typically contain 10%–20% foetal bovine serum (FBS). The use of FBS is associated with several disadvantages, such as batch‐to‐batch variation, safety and ethical concerns. In this study, the suitability of serum‐free media for the cultivation of Theileria‐transformed cell lines was examined. Three commercial serum‐free media (HL‐1, ISF‐1 and Hybridomed DIF 1000) were evaluated for their ability to support growth of the T. annulata A288 cell line. The generation doubling times were recorded for each medium and compared with those obtained with conventional FBS‐containing RPMI‐1640 medium. ISF‐1 gave the shortest generation doubling time, averaging 35.4 ± 2.8 hr, significantly shorter than the 52.2 ± 14.9 hr recorded for the conventional medium (p = .0011). ISF‐1 was subsequently tested with additional T. annulata strains. The doubling time of a Moroccan strain was significantly increased (65.4 ± 15.9 hr) compared with the control (47.7 ± 7.5 hr, p = .0004), whereas an Egyptian strain grew significantly faster in ISF‐1 medium (43.4 ± 6.5 hr vs. 89.3 ± 24.8 hr, p = .0001). The latter strain also showed an improved generation doubling time of 73.7 ± 21.9 hr in an animal origin‐free, serum‐free, protein‐free medium (PFHM II) compared with the control. Out of four South African T. parva strains and a Theileria strain isolated from roan antelope (Hippotragus equinus), only one T. parva strain could be propagated in ISF‐1 medium. The use of serum‐free medium may thus be suitable for some Theileria cell cultures and needs to be evaluated on a case‐by‐case basis. The relevance of Theileria cultivation in serum‐free media for applications such as vaccine development requires further examination.