The Rev protein of equine infectious anaemia virus (EIAV) was shown previously to stimulate the expression of a heterologous CAT reporter gene when the 3' half of the EIAV genome was present downstream in cis. However, computer analysis could not reveal the existence of a stable RNA secondary structure that could be analogous to the Rev-responsive element of other lentiviruses. In the present study, the inhibitory RNA element designated the cis-acting repressing sequence (CRS) has been localized to the centre of the EIAV genome. The inhibition exerted by this element could be overcome by supplying Rev in trans. The ability of the EIAV CRS to function in a heterologous context suggests that it does not require interactions with other viral proteins. Site-directed mutagenesis showed that the various centrally located suboptimal splice sites of the EIAV genome function as CRS and confer Rev-dependence on the CRS-containing transcripts. In addition, the data suggest that in canine Cf2Th cells, which are highly permissive for EIAV replication, CRS prevents nuclear export of CRS-containing transcripts and the supply of Rev relieves this suppression.