Technical information to facilitate bovine blood treatment for optimum lymphocyte flow cytometry analysis is reported. Murine monoclonal antibodies CC8 and CC63 were used to identify phenotypes corresponding to bovine CD4 T cells and CD8 T cells. Blood samples collected in acid citrate dextrose (ACD) enhanced leucocyte subpopulation separation compared with ethylenediamine tetra-acetic acid, heparin and sodium citrate. To preserve bovine blood before immunophenotyping, samples collected in ACD may be kept at 22 degrees C or at 4 degrees C and should be analysed within 32 hours. For isolation of white blood cells, whole blood lysis was faster and gave the same results as Ficoll gradient separation 1.077 and Ficoll gradient separation 1.083. After immunophenotyping, blood could be stored at 4 degrees C if fixed with paraformaldehyde within seven days. Owing to diurnal variations, blood should be collected at a standard time of the day.