Subpopulations of human peripheral blood lymphocytes were studied with single and double surface marker assays. The markers employed were as follows: Fc receptor, using EA [Immunoglobulin(Ig)G-coated bovine red blood cells], mean 21.4%; C3 [3rd component of complement] receptor, using Zy-C3 [C3-coated zymosan particles] mean 14.6%; S[surface]lg, using a fluoresceinated F(ab'')2 anti-F(ab'')2 mean 8.9%; and T [thymus-derived] lymphocytes forming E[erythrocyte]-rosette, mean 69.3%. The proportions of lymphocytes with any given surface marker which also have another surface marker were as follows: EA + Slg/EA = 0.14; EA + Zy-C3/EA = 0.22; Slg + Zy-C3/Slg = 0.83; Zy-C3 + E-rosette/E-rosette = 0.02; EA + E-rosette/E-rosette = 0.06; Slg + E-rosette/E-rosette = 0. The relative percentages of cells which were Slg+EA-, EA+Slg- and EA+Zy-C3- were confirmed by EA rosette depletion via gradient centrifugation. Calculations were made of the theoretical numbers of lymphocytes with triple markers (EA+Slg+Zy-C3+ = 3.0%) and lymphocytes with only a single surface marker (EA+ only = 12.5%, Zy-C3+ only = 4.5%, Slg+ only = 1.5%). A schematic representation of the multiple subpopulations of lymphocytes with various surface markers is presented.