Here we investigate the roles of human testicular orphan receptors, TR2 and TR4, on the gene regulation of the long-terminal repeat of the human immunodeficiency virus type 1 (HIV-LTR). In gel-retardation assays, a palindromic element at the 5'-end of HIV-LTR,5'-AGGGGTCAGATATCCACTGACCTTT-3',showed high affinity to TR2 and TR4 with an equilibrium dissociation constant (Kd) of 1.11 +/- 0.48 (n = 3) and 0.52 +/- 0.12 nM (n = 3), respectively. Interestingly, each half-site of the palindromic element is sufficient to compete with the binding of the labeled palindromic element to TR2 or TR4 with an equilibrium inhibition constant (ki) around 10 nM. However, the transiently expressed TR2 or TR4 in Chinese hamster ovary (CHO) cells or Japanese quail muscle myoblasts (QM7) cells showed no activity in regulating the transcriptional activity of the chloramphenicol acetyltransferase (CAT) reporter gene inserted downstream of the HIV-LTR promoter. Although both TR2 and TR4 showed no effect on CAT activity by itself, our data showed only the TR4 could crosstalk to the chicken ovalbumin upstream protein-transcription factor (COUP-TF1) and thyroid hormone receptor (TR alpha 1), and potentiated the transcriptional activity of HIV-LTR on the CAT reporter gene regulated by COUP-TF1 and TR alpha 1. These results indicate that TR4, but not TR2, may couple to other nuclear receptors in the upregulation of the HIV replication.