We present a procedure for cosmid cloning that allows rapid and efficient cloning of individual DNA fragments of between 32kb and 45kb. By appropriate treatment of the cloning vector, pJB8, we make left-hand and right-hand vector ends that are incapable of self-ligation but which accept dephosphorylated insert DNA fragments. The inserted fragments are generated by partial digestion with MboI or Sau3A and are dephosphorylated to prevent ligation and insertion of non-contiguous fragments. The method eliminates the need to size the insert DNA fragments and prevents formation of clones containing short or multiple inserts. 1μg of target Drosophila DNA gives about 5×105 clones, with an average insert size of 38kb. We also describe a rapid and efficient method for preparing plasmid and cosmid DNA.