The long terminal repeats (LTRs) of equine infectious anemia virus (EIAV) were examined with respect to their ability to function as transcriptional promoters in various cellular environments. Nucleotide sequence analyses of the LTRs derived from two unique proviral clones revealed the requisite consensus transcription and processing signals. One of the proviruses possessed a duplication of a 16-base-pair sequence in the CCAAT box region of the LTR which was absent in the other provirus. To assess its functional activity, each LTR was coupled to the bacterial chloramphenicol acetyltransferase gene and transfected onto various cell lines, including matched cultures of EIAV-infected and uninfected cells. The levels of chloramphenicol acetyltransferase activity directed by the EIAV LTRs were between 250 and 900 times greater in EIAV-infected cells compared with their uninfected counterparts. Thus, EIAV expression appears to be activated by a virus-induced trans-activation phenomenon analogous to that recently shown to amplify expression of certain other lentiviruses.