A continuous-flow technique was developed to isolate and concentrate lymphocytes over Ficoll-Hypaque (FH) density gradients using an automated cell separator (Model CS-3000, Fenwal). Lymphocyte concentrates (LC) containing 6.51 X 10(9) mononuclear cells were obtained by standard leukapheresis techniques. Disposable apheresis kits were modified to allow the LC to be pumped into a separation chamber along with a counter-centrifugal flow of saline, removing the platelets and plasma by elutriation. The remaining cells were underlaid with 300 ml of FH, displacing the lymphocytes into a collection bag, where they were washed and concentrated. Mean leukocyte recovery was 59.2 percent (99.9% lymphocytes, n = 14). The final product contained 6.7 percent of the initial platelets and had a hematocrit of less than 1 percent. In paired studies using split leukocyte concentrates (n = 15), lymphocyte recovery obtained by the automated apheresis technique compared favorably with that obtained by standard manual FH gradients (59.8 +/- 3.4% versus 67.3 +/- 4.2%, p greater than 0.05) and platelet contamination was significantly reduced (2.7 +/- 0.5% versus 26.6 +/- 5.7% residual platelets, p less than 0.001). Equivalent lymphokine-activated killer (LAK) activity was generated from cells isolated by both manual and automated techniques. An automated continuous-flow cell separator can be used for rapid FH isolation of large numbers of lymphocytes, providing a sterile product suitable for human use.