An important role for the Rel/NF-kappaB family of transcription factors in the differentiation process of dendritic cells (DC) and macrophages (MAC) was recently suggested by a number of mouse knockout studies but only little information is available for defined populations of human cells. To investigate the role of individual NF-kappaB proteins [p50, p52, p65 (RelA), RelB] in the differentiation of monocyte-derived cell types we analyzed and compared the expression pattern and DNA binding activity of NF-kappaB members in human monocytes (MO), MO-derived MAC, and MO-derived DC. Constitutive expression of p65 and RelB mRNA was found in MO and no significant regulation was observed during differentiation of MO into MAC or immature DC. Only during lipopolysaccharide-induced terminal differentiation of DC was a marked increase in RelB mRNA detected. In DNA binding assays performed with nuclear extracts from blood MO, p50/p50 homodimers were mainly detected, whereas complexes containing p50/RelB and p50/p65 heterodimers were less abundant. DNA-bound protein complexes containing p50/RelB and p50/p65 increased and additional p65/p65 complexes appeared during differentiation of MO into either MAC or immature DC. A strong increase in complexes containing p50/RelB was observed during terminal differentiation of DC. Therefore, gradual differences in the DNA binding activities of different NF-kappaB homo- and heterodimers correlate with differentiation stages of MO, MAC, and DC and are probably important for the biological role of these cells.