Cis-regulatory elements in the long terminal repeat (LTR) of human foamy virus (HFV) were identified by using LTR mutants to transiently express the chloramphenicol acetyl-transferase gene after co-transfection with an expression plasmid for the virus bel-1 (transactivator) gene. The R-U5 region and an element in the 5' U3 region were found to negatively influence HFV gene expression. The complete BEL-1 responsive region was mapped to extend from nucleotide position -471 to position -93 relative to the start of transcription. Within this region, three elements were identified that in the homologous or a heterologous (SV40) promoter context can, independently and irrespective of their orientation, act as targets for BEL-1. These elements are located between nucleotide positions -413/-378, -361/-291, and -124/93. The target elements do not share obvious sequence homologies. The mechanism of HFV transactivation appears to be novel among the complex retroviruses and is likely to involve, as yet, undiscovered cellular DNA binding factors.