The hydrolysates of nucleic acid, six purine bases and pyrimidine bases (cytosine, uracil, guanine, hypoxanthine, adenine and thymine) were separated and determined by using HPLC. It is discussed how the column and mobile phase affect the separation. The peaks of cytosine and adenine are tailed on ordinary C18 column, and they are very good on BDS-C18 column. The KH2PO4-H3PO4 buffer can be used in separation the hydrolysates of RNA and DNA, and the NaAc-HAc buffer is only used in DNA. In addition, pH value is a very important factor for separation. With pH value of mobile phase increasing, the retention times of guanine, hypoxanthine and thymine were first increased and then decreased, adenine was increased, and cytosine and uracil were almost constant. The chosen mobile phase was 0.1 mol/L KH2PO4-H3PO4 buffer, with a pH value of 4.05. It was detected at UV 260 nm. The determination was completed within 10 min. The RSDs were all less than 3% and the recoveries were in the range of 82%-114%. The method has been applied to the detection of yeast hydrolysates.