The chromatographic behavior and the relationship between the retention and the structure of 16 indole alkaloids were investigated on a mu-Bondapak C18 column at different pH with different volume ratios of 0.02 mol/L KH2PO4-CH3OH as mobile phase and a photodiode array detector (DAD) was used. The experimental results demonstrate that the plots of the logarithm of capacity factor k' of the indole alkaloids are nonlinear in the volume fraction of methanol and the capacity factors increase with increasing pH of buffer solution from 2 to 6 then decrease. The effect of different kinds of buffer solution on k' has also been examined. Phosphate and citrate buffer showed stronger elution strength for various indole alkaloids but acetate buffer was shown to be relatively weak. Increase of phosphate buffer concentration gives a little decrease on k'. These results indicate that the optimized separation of 12 indole alkaloids studied in this work can be carried out isocratically in the system of pH 7.5 phosphate buffer-methanol (30:70) on a mu-Bondapak C18 column.