Cell density-induced growth inhibition of osteosarcoma cells (ROS 17/2.8) results in the shutdown of proliferation-specific histone H4 and H2B genes and the concomitant up-regulation of several osteoblast-related genes. In several respects, this reciprocal regulatory relationship is analogous to the proliferation/differentiation transition stage during development of the bone cell phenotype in normal diploid osteoblasts. Here, we comprehensively analyzed the promoter binding activities interfacing with key regulatory elements in the cell cycle-dependent histone and bone-specific osteocalcin genes. Similarly, we examined factors interacting with a series of general transcription regulatory elements that are present in a broad spectrum of promoters. The results show that histone promoter binding activities HiNF-D, HiNF-P/H4TF-2, H4UA-1, and OCT-1, as well as AP-1 activity, are proliferation dependent. These factors decline coordinately during the cessation of proliferation in both ROS 17/2.8 bone tumor cells and normal diploid osteoblasts. Collective down-regulation of these trans-activating factors occurs in both cell types within the physiological context of constitutive regulation of ubiquitous transcription factors (Sp1, ATF, and CCAAT binding proteins). In addition, during growth inhibition of ROS 17/2.8 cells we observe a complex series of modifications in protein/DNA interactions of the osteocalcin gene. These modifications include both increased and decreased representation of promoter factor complexes occurring at steroid hormone response elements as well as tissue-specific basal promoter sequences. These results demonstrate cell growth regulation of the promoter factors binding to the proliferation-specific histone and tissue-specific osteocalcin genes during the cessation of proliferation.