Human monoclonal antibodies have a plethora of applications, justifying the time and effort towards development of techniques for their efficient production. Attempts at establishing efficient reproducible techniques for activation of lymphocytes in culture have met with moderate success. In this study, human lymphocytes from peripheral blood and bone marrow were immunized in vitro with a T-dependent antigen--bovine gamma globulin. Whole blood, bone marrow and separated mononuclear peripheral blood cells were studied for the potential antibody secretory capabilities. The culture conditions included supplementation with heat treated autologous serum, spent medium from U-266 myeloma cell culture, which is known to contain B cell differentiation factors, and varied antigenic concentrations along with exposure duration. Although there was no appreciable difference in response between whole peripheral blood and whole bone marrow, there is a much better response when compared to isolated cell cultures; especially when culture conditions include antigenic withdrawal and supplementation with conditioned medium. However, lower antigenic concentration was required for whole bone marrow cultures. With optimal in vitro conditions for antigenic stimulation standardized, several options are available for the immortalization of such activated cells to obtain stable human hybridomas of interest.