An enhancer element is located in the U3 portion of exogenous avian retrovirus long terminal repeats (LTRs). A similar element has not been detected in the LTRs of ev-1 and ev-2, two avian endogenous viruses (evs) that normally are not expressed in vivo. Experiments were initiated to determine whether minor nucleotide differences in the U3 region of a previously untested ev that is ubiquitously expressed in vivo (ev-3) might confer enhancer function on the LTR of this provirus. This question was addressed by inserting U3 regions from ev-3 and from ev-1 and/or ev-2 both upstream of the herpesvirus thymidine kinase gene promoter and in place of the major enhancer domains of the Rous sarcoma virus LTR and determining their relative effects on transcription. U3 regions from all evs tested were unable to enhance transcription from the thymidine kinase gene promoter, indicating that nucleotide differences in the ev U3 regions do not affect their relative enhancer function and therefore are unlikely to play a role in their differential expression in vivo. Unexpectedly, however, all ev U3 regions were able to augment transcription in an orientation-independent manner in the ev-Rous sarcoma virus hybrid LTRs. Further experiments conducted to determine why this enhancer activity is not detectable in intact ev LTRs demonstrated that it was not due to removal of repressor sequences in the ev fragments used that might normally be present in intact ev LTRs. The lack of detectable enhancer activity in intact ev LTRs also was not explained by a defect in ev promoters that makes them unresponsive to enhancers in cis. These experiments therefore identify sequences that, although unable to function detectably as enhancers in their natural context, can function efficiently in a heterologous context. Data are discussed in terms of the modularity of enhancer elements and possible interactions between enhancers and promoter-specific sequences.