The enhancer region in the human immunodeficiency virus type 1 (HIV-1) 5'-long terminal repeat (LTR) is very important for viral transcription. This promoter sequence binds both nuclear factor-kappaB and NFAT, two important modulators of HIV-1 gene expression. Previous studies have indicated that the enhancer regions of the different HIV-1 clade LTRs differ in their number of NF-kappaB-binding sites. In this study, we have compared the activation potential of the different HIV-1 clade and HIV-2 LTRs and assessed their interaction with NFAT and NF-kappaB. In T-cell lines and primary CD4(+) T-cells, the results showed that the HIV-1 clade E LTR (with a single NF-kappaB-binding site) was the weakest LTR regardless of the tested activators, whereas the HIV-2 LTR was the most responsive LTR. The clade E enhancer region was also demonstrated to be the weakest enhancer region in transfection experiments with luciferase reporter-based vectors. Electrophoretic mobility shift assays with extracts from activated CD4(+) T-cells indicated that, although NF-kappaB and NFAT bound all enhancers, HIV-1 clade E and HIV-2 LTR enhancers were poor binding targets for these two factors. Weak NFAT binding to clade E enhancers was also confirmed using NFAT1-expressing 293T cells in competition experiments. We have also shown the absence of interaction of NF-kappaB or NFAT with the third NF-kappaB repeat present in clade C. However, the clade C enhancer bound NFAT more efficiently than all other enhancer regions tested. Our results hence demonstrate for the first time that differences in the binding of NF-kappaB and NFAT to the enhancer regions could be responsible for some of the observed variation in HIV-1 clade LTR activation, whereas HIV-2 LTR activation seems mostly independent of these interactions.