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Enhancers are cis-acting activators of transcription from homologous or heterologous promoter elements of viral and cellular genes (see refs 1-6 for reviews). The activity of the simian virus 40 (SV40) (refs 7-9) and immunoglobulin heavy-chain gene (IgH) (refs 10, 11) enhancers has been reproduced to some extent in vitro and appears to be mediated by trans-acting factors both in vitro and in vivo. The SV40 enhancer consists of multiple sequence motifs in two domains, A and B (Fig. 1, see ref. 14): domain B contains GT-I and -II and two TC motifs, of which only TC-II is important for enhancer activity in HeLa cells; and domain A contains the P and the two Sph motifs, the repetition of which generates the sequence 5'-ATGCAAAG-3', similar to the 'octameric' sequence of the IgH enhancer, (Fig. 4i; refs 14, 16), where it is important for enhancing activity. Each SV40 enhancer motif is a binding site for a protein or proteins present in HeLa cell nuclear extracts. Unlike the SV40 enhancer, which is active in HeLa and lymphoid B cells, the IgH enhancer is preferentially active in B cells, suggesting that not all the trans-acting factors necessary for its activity are present in HeLa cells. However, the IgH enhancer can compete with the SV40 enhancer in vitro in HeLa or lymphoid cell extracts and in vivo in B cells. Here we show that both human HeLa and BJA-B lymphoid B-cell nuclear extracts contain proteins that bind to specific, sometimes overlapping, motifs of the SV40 enhancer. Some binding is cell-specific, suggesting that it is not the same set of sequence motifs and proteins that is responsible for the enhancer activity in the two cell types. This is confirmed by our results obtained in vivo with mutated SV40 enhancers.