The present study was conducted to determine the chemotactic effects of different leukocyte diluents, dextrose concentrations associated with or without recombinant human interleukin-8 (rhIL-8), lysed leukocyte concentration, and blood plasma source and concentration (homologous vs. heterologous) on fresh equine leukocyte vitality in vitro in addition to testing the effects of two post-thaw extenders (phosphate buffered saline - PBS - or saline) on vitality and generation of reactive oxygen species (ROS) by frozen leukocytes. These experiments were conducted as part of the development of a cryopreservation protocol for equine leukocytes for use in the treatment of endometritis. Assays were performed using acryl chemotactic chambers incubated at 37 o C. After incubation, leukocytes were counted by flow cytometry. The percent of migrating cells did not differ for different concentrations of dextrose. There was no interaction between leukocyte diluents and dextrose concentrations. An interaction between different plasma types and plasma concentrations was observed. The chemotactic effect of lysed leukocytes was directly related to their concentration. The relative amount of live leukocytes after thawing was greater when incubated in PBS than in saline. In conclusion, dextrose was not a strong chemo-attractant for equine leukocytes while homologous plasma, at concentrations of 10, 30, and 60%, was found to be a strong chemo-attractant. Lysed leukocytes and homologous plasma (10, 30, and 60%) had similar chemotactic effects. Cells diluted in PBS and saline had similar vitality and ROS generation in the post-thaw tests when incubated for 15 minutes.