Two-dimensional agarose gel electrophoresis, psoralen cross-linking, and electron microscopy were used to study the effects of positive supercoiling on fork reversal in isolated replication intermediates of bacterial DNA plasmids. The results obtained demonstrate that the formation of Holliday-like junctions at both forks of a replication bubble creates a topological constraint that prevents further regression of the forks. We propose that this topological locking of replication intermediates provides a biological safety mechanism that protects DNA molecules against extensive fork reversals.