The bovine mixed leukocyte culture (MLC) system offers potential benefit for the study of genetic and immunologic mechanisms in this species. Selected parameters of the bovine MLC have been investigated to assess their influence. The findings indicate that maximal MLC response was present at days 5 and 6 with 2 x 10(5) responder to 2 x 10(5) stimulator cells per well or greater. Serum concentrations over a wide range effectively supported cell growth. More importantly, the source and treatment of serum influenced the level of MLC response. Serum autologous to the responder which was heat treated appeared to provide maximal MLC values compared to other sources examined. Antisera to serologically-defined lymphocyte antigens of the stimulating cell population did not affect the response. One-way MLC determinations were consistent when the stimulating cell population was irradiated at 1,000 Rads or greater. Both freshly obtained lymphocytes and lymphocytes held at room temperature for 18 hr provided good sources of MLC responder and stimulator cells; however, lymphocytes held for 18 hr consistently gave higher counts and stimulation indices than freshly obtained lymphocytes. The effect of contaminating RBCs was found to enhance MLC reactivity at lower concentrations and inhibit MLC reactivity when in excess of 7 X 10(9) RBC per well. Stimulator cells were present in both macrophage enriched and depleted populations suggesting several cell populations possess antigens which lymphocytes recognize. Consideration of these parameters were essential in providing optimal, reproducible results.