A series of Saccharomyces cerevisiae/Escherichia coli λ/plasmid expression vectors have been constructed which allow easy excision of the plasmid sequences from λ. Features of six are described, and two designated λPG15 and λAD5, are characterized in detail. Transcription of cloned sequences is controlled by the alternative promoters, ADH2, PGK, GAL10 and SV40 early, and by the CYC1 transcriptional terminator. Unique EcoRI and XhoI restriction sites in the intervening polylinker make these λ vectors compatible for directional cloning of ‘ZAP’-synthesized cDNAs. Inserted DNAs have been previously shown to have high levels of the genetic activity in both S. cerevisiae and E. coli, allowing these vectors to be used for genetic complementation in both species. Plasmid recovery from the λ vector is mediated by the activity of the cre-encoded enzyme upon lox sequences flanking the plasmid and adjoining the λ arms. The plasmids contain the yeast 2 μm origin and E. coli pBR322 origin, the URA3 or TRP1 yeast selectable markers, and ampicillin-resistance marker in E. coli. The usefulness of the λPG15 and the λAD5 cloning vectors was demonstrated by constructing large Neurospora crassa cDNA libraries. The λPG15–N. crassa library was used to infect purE, purC and trpC mutants of E. coli, and complemented and/or suppressed prototrophic colonies were selected. The flexibility and power of this system for cloning of cDNAs is discussed.