A live, frozen experimental vaccine containing Babesia bovis and Babesia bigemina multiplied in vitro was stored in liquid nitrogen after simultaneous cryopreservation using glycerol as cryoprotectant. The viability of the vaccine was tested by inoculating (subcutaneously) three groups of seven steers each, 2, 12 and 24 h after thawing at 40 degrees C and dilution to obtain a dose of 2 x 10(7) of each organism. All vaccinated cattle developed detectable parasitaemia in thin and/or thick blood smears. No statistically significant differences in the prepatent period were detected amongst the cattle groups (analysis of variance). This prepatent period was 12.3 days for B. bovis and 8.4 days for B. bigemina. Vaccinal organisms derived from in vitro culture systems may replace antigens obtained from in vivo culture to produce vaccine against cattle babesiosis.