Abstract Neutral sucrose gradient sedimentation studies indicate that freezethawing of Escherichia coli B r results in double strand breakage of its DNA. Breaks were detected after one hour of frozen storage at −16°C, resulting in a distinct displacement of the major peak of [ 3H]DNA toward the meniscus. In addition at this time a minor shallow peak of radioactivity toward the meniscus appeared indicating accumulation of some small molecular weight DNA fragments of heterogeneous size. These small DNA fragments seemed to become the major DNA breakage product after 1 1 2 and 4 months of frozen storage. Double strand DNA breakage continued through 8 and 12 months of frozen storage. However, the sedimentation profile became progressively more broad and shallow suggesting that the initial DNA fragments may have reassociated into molecular aggregates of heterogeneous molecular composition.