Immunotherapy utilizing the adoptive transfer of lymphokine-activated killer (LAK) cells in conjunction with recombinant intcrleukin-2 (IL-2) can mediate tumor regression in some patients with advanced cancer. The activation of large numbers of LAK cells was performed in roller bottles in a research laboratory setting and required meticulous aseptic technique, at least one skilled technician per patient and one laminar flow hood pet patient. To reduce the complexity and expense of LAK cell generation for human immunotherapy trials we have developed a closed-system automated procedure using a continuous 'flow blood cell separator. PBL were obtained by standard apheresis techniques. Platelets and plasma were elutriated using counter centrifugal flow of saline in the cell separator machine. The washed PBL were underlaid with Picoll-Hypaque (PH) in the original separation bag, Lymphocytes were then flushed into a collection bag where they were concentrated and washed with 2 liters of saline. Mean recovery from the automated FH technique Was 54.6 ± 4.3% compared. to 62.3 ± 4.0% using manual methods in 50 ml tubes (P > 0.05). Cells were diluted in the collection bag with RPMI 1640 ± 2% human AB serum and could be dispensed in an automated fashion to polyolefin bags via a sample port with lOOO-150'0 U/ml IL-2. After 3-4 days of culture. in 5% CO2 at 37⁰C, activated cells from the bags were harvested and washed in 8 closed system using the continuous flow cell separator. Cell yield from the harvest was 79.2 ± 5.4% in the automated system compared to 64.9 ± 5.0% in the standard procedure using manual harvest of roller bottles (P <0.01). Lytic capacity of the cells against fresh human tumor in a 4 h 51 Cr release assay was equivalent in cells processed either by the automated or the conventional manual, method. The advantages of a closed system include decreased potential for microbial contamination and reduced labor and capital equipment costs. This technique may be easily adapted for use with other cell collection and culture systems.