Jaagsiekte sheep retrovirus (JSRV), enzootic nasal tumor virus (ENTV), and endogenous sheep retroviruses (ESRVs) are highly related sheep betaretroviruses that display different expression profiles in vivo. JSRV and ENTV are expressed in lungs and nasal adenocarcinomas, respectively, while ESRVs are primarily expressed in the reproductive tract of ewes. Evidence suggests that the cell tropism of JSRV, ENTV, and ESRVs is due to the transcriptional specificity of the LTRs. We have previously found several enhancer elements in the JSRV LTR that are important for lung-specific expression, including binding sites for the lung-specific transcription factor HNF-3beta, as well as binding sites for the ubiquitously expressed transcription factors C/EBP and NF-I. In this study, we have aligned the U3 regions of JSRV, ENTV, and several ESRVs in order to compare the transcriptional enhancer elements of JSRV that are conserved or absent in ESRV and ENTV. All three JSRV U3 sequences examined contain two conserved HNF-3 binding sites, while the ENTV and ESRV U3 regions are not predicted to bind this transcription factor. In addition, the C/EBP binding site is interrupted in the ESRV LTRs, but conserved in the ENTV LTRs. Some enhancer elements are conserved between JSRV and ENTV, but a reporter vector carrying the ENTV-1 LTR showed less activity than a JSRV LTR-driven reporter vector in a lung epithelial cell line. These studies support the importance of LTR enhancer elements in the respective tissue specificities of these exogenous and endogenous betaretroviruses.