A high-speed DNA fragment separation system based on an on-line combination of capillary ITP with CZE (CITP-CZE) and using UV detection at 260 nm was developed. A novel CITP-CZE buffer system of pH 6.1 was designed for the separation of ten DNA fragments with sizes ranging from 100 to 1000 bp. An effect of underivatized alpha-, beta- and gamma-cyclodextrins on the resolution of DNA fragments in the CZE step of the CITP-CZE combination was systematically investigated. Methylhydroxyethylcellulose present in the BGE was used to eliminate the EOF. DNA ladder fragments were separated within 10 min with LODs in the range of 1-5 ng/mu L (S/N = 3). The RSDs of the migration time and peak area of individual DNA fragments were in the range of 1-3 and 3-9%, respectively. The developed CITP-CZE system was further applied to the analysis of digest plasmid DNA samples.