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AIM: To improve the preparation of adherent lymphokine-activated killer (A-LAK) cells and to study the effects ofcryopreservation and phenylacetate (PA) on biologicalcharacters of A-LAK cells.METHODS: A-LAK cells were obtained from peripheral bloodmononuclear cells (PBMCs) of the patients withhepatocellular caminoma (HCC) by using L-phenylalaninemethyl ester ( PME ) to deplete immunosuppressivermonocytes. Proliferative activity of SMMC7721 cell line aftertreatment with phenylacetate (PA) was observed. A-LAKcells were treated with the supernatant of SMMC7721 cellsthat had been pretreated with PA. The changes ofproliferation, cytotoxicity and phenotype of A-LAK cellswere investigated after cryopreservstion.RESULTS: The expansion of A-LAK cells (96.79± 69.10 foldson Day 14 ) was significantly higher then that of non-adherent LAK (NA-LAK) cells (22.77 ± 13.20) as well asconventional LAK cells (4. 64 ± 0. 91 ). PA significantlysuppressed the growth of SMMC7721 cells, and the inhibitorratio was 46 %. rite supernatant of cultured tumor cellsintensively suppressed the proliferation and cytotoxicity ofA-LAK cells, but the suppressive effect of the supernatantwas previously decreased after treatment with PA.Impairments in proliferation and cytotoxicity of A-LAK cellsimmediately after thawing of cryopreservation and recoveryafter reincubation with IL-2 were observed. The cytotoxicltyof thawed A-LAK cells on Day 5 was significantly higher thanthat of fresh A-LAK before freezing (54.8 ± 10.2 % vs 40.5 ±6. 4 %). No significant change in the percentage oflymphocyte subsets was identified in frozen A-LAK cells ascompared with that in the fresh control cells.CONCLUSION: A-LAK cells can he simply prepared by usingPME, and showed a synergistic anti-tumor effect with thecombination of PA. Cryopreseration can increase theimmunoactivities of A-LAK cells from the patients withhepatocellular caminoma .