In the present investigation the antiproliferative activity of thirteen derivatives of betulinic acid and betulin was tested against five different tumor cell lines. The toxicity against normal human fibroblasts (WWO70327) and the mode of cell death on HT-29 (colon cancer) as well as caspase activity induced by the most active compounds, 9 (3-O-chloroacetylbetulinic acid) and 15 (28-O-chloroacetylbetulin) were determined. Investigated derivatives exerted a dose dependent antiproliferative action at micromolar concentrations toward target tumor cell lines. Treatment of HT-29 cells for 24 h with 9 and 15 induced apoptosis, as observed by dye exclusion test (trypan blue) and confirmed by the appearance of a typical ladder pattern in the DNA fragmentation assay.