We describe a simple method for isolation of high-molecular-weight (high-mol-wt, > 800 kilobases) DNA from tumor cell lines frozen without dimethylsulfoxide (DMSO) or other protective agents. This method should be applicable to most frozen tissues and useful for molecular genetic studies such as field-inversion gel electrophoresis (FIGE), or cloning procedures that require access to intact, high-mol-wt DNA.