Lambda/plasmid hybrid vectors have been previously constructed in which the plasmid sequences are separated from flanking lambda arms by lox sites. The lox sequence is the substrate of Cre-mediated site-specific recombination, allowing easy excision of plasmid sequences (automatic subcloning). We have developed a simple procedure to construct other such lambda hybrid vectors using in vivo cre/lox-mediated recombination to exchange new plasmids for plasmids previously incorporated into lambda/plasmid hybrids. Because hybrid vectors both with and without lacZ alpha plasmid sequences are available, producing either blue or clear plaques, respectively, the new lambda hybrid vectors can be distinguished from the parental hybrids by blue/clear plaque screening. This procedure has been successfully used to construct ten hybrid vectors. It generates new lambda/plasmid hybrid vectors, without ligation or lambda packaging, which retain the property of automatic subcloning.