Abundant human retroposons of the Alu family produce few RNA polymerase III (RPIII)-dependent transcripts in vivo. This suggests that either the bulk of the repeats has no proper promoter elements or that transcription of Alu by RPIII is repressed. In this study, we analyzed complexes formed by human nuclear proteins with the Alu B-box and with an adjacent downstream sequence (DB-sequence). Four complexes (C1-C4) were detected and two of them (C2 and C3) were found to be induced by different proteins. C3 formation was found to be sensitive to minor sequence variation within the Alu DB-sequence. The C2 complex is specifically repressed by the competing VA1 B-box oligonucleotide and was found to be very stable. In addition, it is downregulated in human cells transformed by adenovirus 5. This is consistent with a view that the C2 complex is formed by a protein (designated as ACR1) that is different from TFIIIC2. The ACR1 protein may be involved in the modulation of Alu transcription in vivo by interfering or cooperating with TFIIIC2. A similar complex is detected with the efficiently transcribed adenovirus VA1 RNA gene B-box. We compared the affinity of complexes formed by ACR1 with Alu and VA1 B-boxes. It was found that both B-boxes bind ACR1 with equal affinity with a dissociation constant of about 2 nM. However, DB-sequences in Alu and VA1 promoters are non-homologous, and C3/C4 complexes are found to be formed with Alu DB, but not formed with VA1 DB sequences. The Alu-specific protein forming C3 (named as ACR2) may cooperate with ACR1 in selective repression of RPIII-dependent Alu transcription in vivo.