A sensitive procedure employing limiting dilution of activated lymphocytes and 51Cr release from highly labeled target cells was used to derive minimal estimates of the absolute frequency of cytotoxic T lymphocytes (CTL) present in a population of mouse lymphocytes activated to alloantigens of the major histocompatibility complex in bulk mixed lymphocyte cultures. From this figure (0.7-1.2%), the maximal rate of target cell killing could be calculated to be approximately 4 targets/CTL/hour.