The expression of the Arabidopsis thaliana kinesin-like protein 1 (AtKP1) gene is restricted to tender tissues. We used a 5'-deletion assay to identify and characterize the regulatory regions controlling tissue-specific AtKP1 expression. Multiple enhancer regions, located 470- and 2,808-bp upstream of the translational start codon, were critical for activation, while a silencer region located at -2,987 to -2,808 (A + T = 71%) was required for repression. Within this 180-bp fragment, a 43-bp element (termed KPRE, A + T = 58%) mediated repression of the CaMV35S promoter by using a gain-of-function approach that was orientation-dependent in leaves and orientation-independent in roots. Electrophoretic mobility shift assay (EMSA) showed that the GAGAAATT octamer (corresponding to neucleotides -2,908 - -2,900) in KPRE was the core negative regulatory motif for interacting with DNA-binding proteins in leaves and roots. However, using a second gain-of-function experiment with KPRE fused to CaMV35S, we found that the mutant negatively affected transcription in transgenic leaves and positively affected transcription in transgenic roots. This indicated that these two modes mediate repressive regulation in leaves and roots, respectively. The EMSA experiment using different mutant KPRE as probes confirmed that two distinct sets of proteins bound to KPRE at an overlapping site AGAAAT in the leaf. Taken together, these data suggest that two different modes control the negatively transcriptional regulation of KPRE in leaves and roots, and provide new insight into the mechanism of transcriptional repression of A/T-rich sequences in higher plants.