A set of Epstein-Barr virus (EBV) episomal expression vectors, incorporating either the Rous sarcoma virus 3' long terminal repeat or the human metallothionein IIA gene promoter, were constructed. The transcriptional cassettes encompassed by these vectors were designed to permit both antisense and sense RNA transcription. A novel methodology was developed for directional cDNA cloning using an oligodeoxyribonucleotide adapter; the EBV episomal vectors alternatively enabled the insertion of cDNA segments in antisense or sense orientations. We propose a strategy for random antisense RNA mutagenesis exploiting this vector system and a method for episome-based directional antisense cDNA cloning and expression, permitting the rapid identification of genes mediating selectable cellular functions.