Mononuclear cells from the blood of healthy normal humans were kept in cultures under nonstimulating conditions for 16 [14C]thymidi'ne to 0.2 ml of cell supension in microtiter plates which were harvested and assayed for radioactivity 12-16 hr later, according to standard procedures. Cells were washed two or three times in medium containing 5% fetal calf serum And 15 mM Na azide and then were incubated with 1:25 diluted fluorescein isothiocyanate (FITC)-labeled specific rabbit anti-human [B2-m antiserum (Dakopatts, code F112, Dakopatts, Copenhagen) or with a 1:8 dilution of a nonlabeled broadly reacting anti-VJLA antiserum including the specificity Bw4. The serum "Buffo" from the International Serum Bank in Torino was kindly provided by J. Stocker (Basel). After a 45-min incubation, the cells were processed for quantitative immunofluorescence in the fluorescence-activated cell sorter (FACS II, Becton-Dickinson) or were washed three times and incubated with the second layer, which was a 1:50 dilution of FITC-conjugated goat IgG anti-human Fc antibodies (a kind gift of L. Forni, Basel), for a further 30 min.Immunoglobulin-bearing cells were labeled by using FITC-conjugated rabbit IgG anti-human IgM antibodies (Dakopatts, code.F1090) or swine anti-human Ig (polyvalent) from Dakopatts (F2190). Rabbits were immunized at 2-week intervals by two injections of human brain tissue (obtained from autopsies) suspended in complete Freund's adjuvant. After 1 month they received approximately 107 purified human peripheral T lymphocytes intravenously, and serum was harvested 2 weeks later. The antibodies reacted with close to 90% of the blood lymphocytes in a complement-dependent cytotoxicity test and were used unabsorbed in the present study. Cells to be labeled were incubated with a 1:10 dilution of the serum for 45 min at 4°C, washed, and subsequently stained with a 1:50 dilution of FITC-conjugated swine anti-rabbit Ig (Dakopatts,