The two-color method originally described by Van Rood et al. (Van Rood, J. J., A. Van Leeuwen, and J. S. Ploen. 1976. Simultaneous detection of two cell populations by two-color fluorescence and application to the recognition of B-cell determinants. Nature (Lond.). 262: 795-797) for the typing of homologous leukocytic antibodies, D-region was used for the detection of antilymphocyte antibody (ALA) in systemic lupus erythematosus. In this method, surface immunoglobulin-bearing cells were identified with fluorescein isothiocyanate-labeled anti-immunoglobulin and nuclei of killed cells were stained with ethidium bromide. Therefore, cell type (T or B) of the target cells can be identified without fractionating them. ALA was detected in 87% of lupus sera and had a preferential reactivity with T cells. Its major immunoglobulin class was shown to be immunoglobulin (Ig)M. The subspecificity of ALA was further analyzed using fractionated T-cell subsets as target cells. When T lymphocytes were separated into Fc receptor-bearing (Tgamma) and lacking (Tgamma[-]) cells, 64% of ALA showed preferential reactivity with Tgamma cells and 14% with Tgamma(-) cells. The remainder had no selective reactivity against Tgamma or Tgamma(-) cells. Tgamma cells were shown to have suppressor activity, whereas Tgamma(-) cells were indicated to contain helper cells. The above finding was in agreement with the observation that treatment of T cells with ALA that preferentially react with Tgamma cells considerably enhanced immunoglobulin synthesis in vitro, whereas treatment of T cells with ALA reactive with Tgamma(-) cells clearly suppressed the formation of immunoglobulins. Treatment of ALA with no selective reactivity showed variable effects on in vitro immunoglobulin synthesis. These results indicate that ALA in lupus have heterogeneous specificities against human T-cell subsets.