Live frozen vaccines containing Babesia bovis, Babesia bigemina or Anaplasma centrale were prepared using glycerol as cryoprotectant and stored in liquid nitrogen. The viability of the vaccines was tested inoculating calves 1 h (n = 12), 2 h (n = 12), 12 h (n = 6) and 24 h (n = 6) after thawing. Babesia bovis and A. centrale were detected in thin and/or thick blood smears in all vaccinated calves; however, 1 of 12 calves inoculated 1 h after thawing and 3 of 6 calves inoculated 24 h after thawing did not develop a B. bigemina parasitaemia. The longer post-thawing durability of frozen vaccines cryoprotected with glycerol compared with those cryoprotected with dimethyl sulfoxide, presented by other authors, will extend their use under field conditions.