Purpose of this study was to establish an effective method in vitro to proliferate natural killer T (NKT) cells from umbilical cord blood (UCB) and peripheral blood (PB), and to study their different phenotype. Mononuclear cells (MNC) from UCB and PB were cultured in the presence of IL-2 (100 U/ml), with or without alpha-Galcer. TCR Valpha24 Vbeta11 double positive natural killer T-cells (NKT cells) and their other phenotypes were determined by flow cytometry. The results showed that after expansion for 7 days, TCRValphabeta(+) NKT cells from UCB-MNCs increased by (8.74 +/- 4.37) x 10(2) times as much, but most of them did not express NK1.1 and its TCR Vbeta11(+) was higher than TCR Valpha24(+). After expansion for 14 days, TCR Valphabeta(+) NKT cells from PB-MNCs increased by (3.72 +/- 2.01) x 10(2) times, the expression of NK1.1 was high and its TCR Vbeta11(+) was almost equal to TCR Valpha24(+). It is concluded that human TCR Valpha24 Vbeta11 double positive NKT cells can expand by addition of alpha-Galcer. The proliferation efficiency in UCB-MNCs is greater than that in PB-MNCs. Most of the UCB-NKT is NK1.1(-), while the PB-NKT is NK1.1(+), a new subset of NKT cells.