The humoral immune response propels the production of a diversified pool of antibodies with high affinity and selectivity for the eliciting antigen. Their isolation entails either B-cell cloning or the linking of authentic pairs of variable region genes encoding them. We hypothesized that targeted RNA trans-splicing (TS) inside the B-cell nucleus could be harnessed as a novel means to link both variable region genes and reconstitute genuine immune B-cell specificities. This could be accomplished by a special targeting gene harboring a peptide linker exon flanked by sequences capable of targeting both heavy (HC) and light chain (LC) transcripts. Following sequential trans-splicing reactions, the resulting RNA in each cell would encode the two variable regions, joined by the peptide linker. In this study, we examined genetic components and configurations required for the separate trans-splicing steps and for the combined two-step reactions. Using a model antibody, we show that in transiently transfected cells, we can target variable region exons through both their acceptor and donor splice sites, precisely joining an exon encoding a synthetic linker and the complementary variable region so as to form a single-chain Fv. We also demonstrate the accurate formation of single-chain Fv transcript as a result of trans-splicing of RNA synthesized from two chromosomal genes expressed by a stably transfected B-cell hybridoma. Our attempts to link the two variable region genes via a synthetic linker exon through sequential trans-splicing events were only successful with regard to both ends of the linker and to the 3' end of the light chain, but repeatedly resulted in a deletion at the 5' end of the joined heavy chain transcript. The implications of our findings on the potential application of trans-splicing for the isolation of useful antibodies are discussed.