Expression of the rat luteinizing hormone receptor (rLHR) is characterized by a dynamic response to a variety of hormonal stimuli. In addition to activation, the pattern of rLHR expression is also modulated by repression. In this report, an upstream initiator-like element (UInr-lE), CTCACTCTAA, of which the CTC direct repeat motif (CTCACTC) is conserved in the rat, mouse and human, was identified as a suppressor element. Disruption of the element resulted in a two-fold enhancement of promoter activity in the LHR-expressing murine Leydig tumor cells (mLTC-1). The sequences of the two major initiators (Inr), Inr3 and Inr4 of the rLHR core promoter are similar to UInr-lE, and competed efficiently with UInr-lE in the formation of specific protein complexes, suggesting that the same proteins interact with both UInr-lE and the Inrs in vivo. The Inrs are necessary for full promoter activity because a mutant promoter lacking Inrs showed a 70% reduction in activity. UInr-lE also further suppressed the activity of a mutant promoter lacking Inrs. UInr-lE interacted with TFII-I and an unidentified nuclear protein. However, dominant-negative inhibition experiments using p70 indicated that TFII-I positively regulates LHR promoter activity through UInr-lE and Inrs, suggesting that TFII-I can compromise the suppression of promoter activity mediated by UInr-lE. UInr-lE also showed binding properties distinct from that of the upstream initiator-like suppressor element (URE: CACTCTCC) of rat and human dynorphin (rDyn) promoters. Transfection assays using mutated promoters indicate that the suppression of rLHR promoter activity could be regulated via specific interactions between UInr-lE and trans-acting factors.