The effect of gelation conditions on the structure and resolving properties of agarose-based DNA sequencing gels were studied. Gels that were formed by rapid cooling gave separation patterns with sharp, well-defined bands, and resolved base repeats that were not separated on gels which were allowed to cool slowly. Analyses by electron microscopy indicated that the rapidly cooled gels have a homogeneous pore structure with a smaller average pore size than gels that were cooled slowly. The two types of gels also differ in optical clarity, and have significant differences in their melting characteristics as measured by differential scanning calorimetry.