CD11c integrin expression is restricted to myeloid cells and activated B lymphocytes, mainly through the collaborative action of Sp1 and members of the AP-1 and C/EBP transcription factor families on the proximal region of the CD11c gene promoter. While analyzing the role of an initiator-like sequence at the major transcriptional start site, an inverted consensus GGAA Ets binding site was identified as a negative regulatory element whose disruption increases the activity of the CD11c promoter. The GGAA element was specifically recognized by PU.1 in THP-1 monocytic cells and by PU.1 and GABP-related proteins in U937 promonocytic cells. Mutational analysis indicated that PU.1 recognition depends not only on the GGAA consensus element but also on flanking sequences. The functional relevance of PU.1 binding was assayed in transactivation experiments in HeLa cells, where PU.1 co-expression led to a significant decrease in the activity of the CD11c promoter, demonstrating that PU.1 inhibits the activity of the CD11c promoter through a PU.1 binding site located at the major transcriptional start site (PU1-5). The inhibitory action of PU.1 on CD11c is in contrast with its positive regulatory effect on the CD11b and CD18 integrin gene promoters, which might contribute to the differentially regulated expression of CD11b/CD18 and CD11c/CD18 during monocyte extravasation and terminal maturation. In addition, since PU.1 transcriptional activity correlates with macrophage proliferation, PU.1 might modulate CD11c gene transcription according to the proliferative state of the cell.