We demonstrate a novel method for stretching a long DNA molecule in agarose gel with alternating current (AC) electric fields. The molecular motion of a long DNA (T4 DNA; 165.6 kb) in agarose gel was studied using fluorescence microscopy. The effects of a wide range of field frequencies, field strengths, and gel concentrations were investigated. Stretching was only observed in the AC field when a frequency of approximately 10 Hz was used. The maximal length of the stretched DNA had the longest value when a field strength of 200 to 400 V/cm was used. Stretching was not sensitive to a range of agarose gel concentrations from 0.5 to 3%. Together, these experiments indicate that the optimal conditions for stretching long DNA in an AC electric field are a frequency of 10 Hz with a field strength of 200 V/cm and a gel concentration of 1% agarose. Using these conditions, we were able to successfully stretch Saccharomyces cerevisiae chromosomal DNA molecules (225-2,200 kb). These results may aid in the development of a novel method to stretch much longer DNA, such as human chromosomal DNA, and may contribute to the analysis of a single chromosomal DNA from a single cell.