Human adenovirus E1A oncoprotein activates or represses transcription from a variety of viral and cellular promoters by several complex mechanisms. The E1A products, 289R and 243R, have differential effects on transcription directed by the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR). Previous reports indicate that repression of HIV-1 LTR-directed gene expression by E1A 243R is mediated through the kappa B enhancer elements located between nucleotides -105 and -82 relative to the transcription initiation start site (+1). Results from this study suggest a novel mechanism for transcriptional repression of the HIV-1 LTR by E1A 243R that is enhancer-independent and that is mediated through basal HIV-1 promoter elements. Transient expression assays, in which 5'-truncated or site-directed mutant HIV-1 LTR-CAT reporters were tested for their response to repression mediated by wild-type or mutant 243R, demonstrate that LTR sequences upstream of -31 relative to the transcription initiation start site (+1) and inclusive of the enhancer elements are dispensable for 243R-mediated repression. The ability of 243R to repress HIV-1 basal promoter activity requires both an intact N-terminus of E1A 243R and the TATA element within the HIV-1 promoter. These results support a novel mechanism for E1A 243R-induced transcriptional repression that is enhancer-independent and that targets directly the general transcription machinery.