A highly specific anti-T cell antiserum (ATCS) was developed by extensively absorbing a goat anti-human thymocyte antiserum with human red cells and buffy coat cells from a patient with chronic lymphocytic leukemia. The ATCS was used in an in vitro cytotoxicity assay for detecting human thymus-derived (T) peripheral blood lymphocytes. A fluorescent cell sorter provided separated populations of normal immunoglobulin (Ig)-bearing and non-Ig-bearing lymphocytes on which to directly test the sensitivity and specificity of the ATCS. The antiserum killed almost 100% of non-Ig-bearing lymphocytes but less than 10% of Ig-bearing (B) lymphocytes and monocytes. Moreover, ATCS was able to abrogate the response to phytohemagglutinin stimulation, a T cell response. In further studies, Ficoll-purified peripheral blood cells from 12 normal donors were examined for the per cent Ig-bearing cells, monocytes and cells killed by ATCS. A mean of 21% of these cells were B lymphocytes, 17% were monocytes, and 65% were T lymphocytes, thus accounting for 100% of the cells.