Arecoline inhibits hepatoma cell colony growth in soft agar. The growth of HA22T/VGH cells in soft agar was measured in 35 mm diameter dishes with a lower layer of 0.7% agar solution and an upper layer of 0.35% agar solution in which 1 × 105 cells were resuspended. The soft agar was covered with culture medium alone or containing the indicated concentration of arecoline. After 3 weeks, colonies larger than 0.1 mm in diameter were scored. (A) Representative photomicrographs of soft agar colonies at 200× magnification. The black arrows indicate colonies. (B) Number of colonies in the presence or absence of arecoline. The results are the mean ± S.D. for three independent experiments. *: p < 0.05; #: p < 0.001 as compared to the untreated control.