Adeno-associated virus type 2 (AAV-2), a human parvovirus which is apathogenic in adults, inhibits replication and gene expression of human immunodeficiency virus type 1 (HIV-1) in human cells. The rep gene of AAV-2, which was shown earlier to be sufficient for this negative interference, also down-regulated the expression of heterologous sequences driven by the long terminal repeat (LTR) of HIV-1. This effect was observed in the absence of the HIV-1 transactivator Tat, i.e., at basal levels of LTR-driven transcription. In this work, we studied the involvement of functional subsequences of the HIV-1 LTR in rep-mediated inhibition in the absence of Tat. Mutated LTRs driving an indicator gene (cat) were cointroduced into human SW480 cells together with rep alone or with double-stranded DNA fragments or RNA containing sequences of the HIV-1 LTR. The results indicate that rep strongly enhances the function of negative regulatory elements of the LTR. In addition, the experiments revealed a transcribed sequence element located within the TAR-coding sequence termed AHH(H) (AAV-HIV homology element derived from HIV-1) which is involved in rep-mediated inhibition. The AHH(H) element is also involved in down-regulation of basal expression levels in the absence of rep, suggesting that AHH(H) also contributes to negative regulatory functions of the LTR of HIV-1. In contrast, positive regulatory elements of the HIV-1 LTR such as the NFκB and SP1 binding sites have no significant influence on the rep-mediated inhibition.