The ex vivo expansion of antigen-specific cytotoxic T lymphocyte (CTL) lines is being developed for immunotherapy of viral infections and cancer and is critically dependent on the precise cell expansion and stimulation conditions. In this article, we investigate medium requirements for the development of HIV-specific CTL in cell lines generated from the peripheral blood of seven asymptomatic HIV-infected individuals. We find that HIV specific CTL do not readily develop in the serum-free medium AIM V but do develop if the medium is supplemented with 1% plasma or serum. T cell lines with antigen-specific cytolytic activity express more cell-surface CD57 than do cell lines grown in the absence of serum or plasma. Three sources of serum (human autologous, human AB, or fetal calf) are comparable. Human plasma is somewhat less effective than serum from an identical source.