Human monocyte to macrophage differentiation is accompanied by pronounced phenotypical changes and generally proceeds in the absence of proliferation. The molecular events governing this process are poorly understood. Here, we studied the regulation of the macrophage-specific chitotriosidase (CHIT1) gene promoter to gain insights into the mechanisms of transcriptional control during the differentiation of human blood monocytes into macrophages. We used transient transfections to define a cell type-specific minimal promoter that was mainly dependent on a proximal C/EBP motif that bound multiple C/EBP factors in gel shift assays. In depth analysis of occupied promoter elements using in vivo footprinting and chromatin immunoprecipitation analyses demonstrated the differentiation-associated recruitment of C/EBPbeta and PU.1 at the proximal promoter in parallel with CHIT1 mRNA induction. Notably, the induction of C/EBPbeta promoter binding strongly correlated with increased nuclear levels of Thr-235-phosphorylated C/EBPbeta protein during the differentiation process, whereas C/EBPbeta mRNA and total protein expression remained relatively stable. Our data suggest an important constitutive gene regulatory function for C/EBPbeta in differentiated macrophages but not in human blood monocytes.