We demonstrate an alternative method for the generation of random subclones for large-scale shotgun human DNA sequencing projects. Miniprep DNA from a human cosmid clone was partially digested with CviJI, size-fractionated by agarose gel electrophoresis and cloned into bacteriophage M13. A library consisting of 10(5) subclones of 1.1 kb average size was recovered from one gel fraction containing approximately 300 ng of partially digested DNA. DNA sequences from an initial 103 subclones demonstrate that 100 of the subclones cover 90% of the cosmid, which is close to the 92% expected if the subclones were generated at random. DNA sequences from three of the subclones did not match the cosmid sequences, establishing that miniprep DNA can be used for library construction with little concern for contamination with genomic E. coli DNA. The use of CviJI to generate random DNA fragments thus offers a simple alternative to other commonly used fragmentation methods, such as shearing or sonication, for the generation of random sublibraries for large-scale human shotgun DNA sequencing projects.