Discontinuous bovine serum albumin gradients were used to fractionate peripheral blood leukocytes from bovine leukemia virus (BLV)-free and BLV-infected cows. The release of infectious BLV and spontaneous incorporation of [3H]thymidine were not properties of density gradient-fractionated leukocytes from a BLV-free cow. When leukocytes from BLV-infected cattle were fractionated, B lymphocytes which spontaneously incorporated [3H]thymidine could be separated as a distinct subpopulation from B lymphocytes which replicated infectious BLV. Density gradient fractionation of leukocytes from a cow with lymphosarcoma is also reported. A fall in lymphocyte count at the time of tumor development is attributed to the loss of B lymphocytes which spontaneously incorporate [3H]thymidine.