The aim of the study was to assess the effect of selected isolation methods on the viability and metabolism of bovine leukocytes. The cells were isolated using a Ficoll 1077, Histopaque 1083 gradient and osmotic shock method, and Ficoll or Histopaque with osmotic shock. Evaluation were made of the total number of cells, viability after isolation and in 24h culture on RPMI 1640 medium and metabolism with NBT reduction assay. Microscopic and cytometric evaluation of the leukocytes revealed that the isolation methods applied had an influence on their number and viability. Based on the results it can be concluded that isolation methods of cells in a Histopaque or Ficoll yield highly pure cell fractions with high viability.