The immunoglobulin heavy chain intron enhancer (Emu) not only stimulates transcription but also V(D)J recombination of chromosomally integrated recombination substrates. We aimed at reproducing this effect in recombination competent cells by transient transfection of extrachromosomal substrates. These we prepared by interposing between the recombination signal sequences (RSS) of the plasmid pBlueRec various fragments, including Emu, possibly affecting V(D)J recombination. Our work shows that sequences inserted between RSS 23 and RSS 12, with distances from their proximal ends of 26 and 284 bp respectively, can markedly affect the frequency of V(D)J recombination. We report that the entire Emu, the Emu core as well as its flanking 5' and 3' matrix associated regions (5' and 3' MARs) upregulate V(D)J recombination while the downstream section of the 3' MAR of Emu does not. Also, prokaryotic sequences markedly suppress V(D)J recombination. This confirms previous results obtained with chromosomally integrated substrates, except for the finding that the full length 3' MAR of Emu stimulates V(D)J recombination in an episomal but not in a chromosomal context. The fact that other MARs do not share this activity suggests that the effect is no mediated through attachment of the recombination substrate to a nuclear matrix-associated recombination complex but through cis-activation. The presence of a 26 bp A-T-rich sequence motif in the 5' and 3' MARs of Emu and in all of the other upregulating fragments investigated, leads us to propose that the motif represents a novel recombinational enhancer element distinct from those constituting the Emu core.