A replaceable polymer matrix, based on the novel monomer N-hydroxyethylacrylamide (HEA), has been synthesized for application in DNA separation by microchannel electrophoresis. The monomer was found by micellar electrokinetic chromatography analysis of monomer partitioning between water and 1-octanol to be more hydrophilic than acrylamide and N,N-dimethylacrylamide. Polymers were synthesized by free radical polymerization in aqueous solution. The weight-average molar mass of purified polymer was characterized by tandem gel permeation chromatography-multiangle laser light scattering. The steady-shear rheological behavior of the novel DNA sequencing matrix was also characterized, and it was found that the viscosity of the novel matrix decreases by more than 2 orders of magnitude as the shear rate is increased from 0.1 to 1000 s(-1). Moreover, in the shear-thinning region, the rate of change of matrix viscosity with shear rate increases with increasing polymer concentration. Poly-N-hydroxyethylacrylamide (PHEA) exhibits good capillary-coating ability, via adsorption from aqueous solution, efficiently suppressing electroosmotic flow (EOF) in a manner comparable to that of poly-N,N-dimethylacrylamide. Under DNA sequencing conditions, adsorptive PHEA coatings proved to be stable and to maintain negligible EOF for over 600 h of electrophoresis. Resolution of DNA sequencing fragments, particularly fragments > 500 bases, in PHEA matrices generally improves with increasing polymer concentration and decreasing electric field strength. When PHEA is used both as a separation matrix and as a dynamic coating in bare silica capillaries, the matrix can resolve over 620 bases of contiguous DNA sequence within 3 h. These results demonstrate the good potential of PHEA matrices for high-throughput DNA analysis by microchannel electrophoresis.