Simian foamy viruses, members of the spumavirus subfamily of retroviruses, are found in a variety of nonhuman primates and, as yet, remain to be characterized with respect to genetic structure and regulation of viral gene expression. The genome of simian foamy virus type 1 (SFV-1), an isolate from rhesus macaques, has been molecularly cloned, and the role of the viral long terminal repeat (LTR) in transcriptional control has been investigated. The SFV-1 LTR is 1,621 base pairs long, and sequence comparisons with human foamy virus revealed a pattern of clustered homology. A cap site in the LTR was identified by analysis of SFV-1 transcripts in infected cells. Transient expression assays in cell lines representing several species and different cell types showed that the SFV-1 LTR has low basal activity in uninfected cells, whereas LTR-directed expression is greatly increased in cells infected with SFV-1. This transactivation is mediated by a mechanism involving increases in steady-state levels of viral transcripts. Thus, the SFV-1 genome encodes a transactivator that functions on the LTR at the transcriptional level.