The myeloid integrin CD11b is expressed selectively on the surface of mature macrophages, monocytes, neutrophils, and natural killer cells. Lineage-specific expression is controlled at the level of mRNA transcription. Recent isolation of the CD11b promoter shows that 92 base pairs (bp) of 5'-flanking DNA are sufficient to direct myeloid-specific expression of a reporter gene. To characterize regulatory sequences important for promoter activity, we performed linker scanning analysis of the 92-bp CD11b promoter and demonstrate that a sequence at bp -60 is essential for CD11b promoter activity. We show that this sequence binds the transcription factor Sp1 in vitro and in vivo. In vivo the Sp1 site is bound only in myeloid (U937) cells, not in cervical carcinoma (HeLa) cells. In addition, the macrophage transcription factor PU.1 binds the CD11b promoter in vitro and in vivo close to the Sp1 site. We propose a model in which binding of a myeloid-specific factor (PU.1) allows a general factor (Sp1) to bind in a tissue-specific fashion thereby contributing to the myeloid-specific expression of CD11b.