An evaluation of some system variables for the suspension-culture method of in vitro Babesia bovis cultivation has identified some variables as critical and suggested optimum values for others. Growth of Babesia was not affected by siliconizing the culture vessels or including HEPES buffer in the culture medium. THe culture pH, however, markedly influenced the growth rate. When supplemented with a large amount of serum, even Hanks's balanced salt solution was an adequate medium to support growth of B bovis, but growth improved with increasing medium complexity. A comparison of growth in the complex mammalian tissue culture mediums RPMI-1640, medium 199, and NCTC-135 showed no significant differences. Heparinized plasma (plasma with added heparin ) could not be substituted for serum. While serum could be stored by either freezing or refrigeration at 4 C for up to a week RBC rapidly lost their ability to support growth of B bovis when stored at 4 C. Preliminary experiments with the gaseous environment indicated that CO2 is required and that the optimum oxygen tension was near the normal atmospheric level of about 20%.