Fifty-one blood samples of carrier horses from Theileria equi-endemic localities in South Africa were used for two different methods of in vitro culture initiation of T. equi parasites. Cultures were initiated either in a oxygen-reduced gas mixture or in a 5% CO2-in-air atmosphere in combination with L-cysteine-supplemented culture medium. Out of the 51 blood samples, 43 and 42 cultures, respectively, became culture positive. A possible explanation for this observation is proposed.