We introduce two new methods for target cloning of DNA fragments corresponding to spots on the two-dimensional profile of restriction landmark genomic scanning (RLGS). One is a restriction trapper-based method and the other is a polymerase chain reaction (PCR) mediated method. Both are designed to select the target DNA fragments from a large amount of unlabeled background DNA fragments in the RLGS gel which produce background clones. The restriction trapper method is simple, with a cloning efficiency that is not biased by the length of the target DNA nor by its GC content. On the other hand, the PCR-mediated method is efficient for cloning DNA fragments from a small amount of starting materials. These methods provide us with powerful tools for isolating DNA clones identified by the RLGS system as interesting spots. This paper reports the precise protocols of these methods and discusses their application and usefulness.