Osteoblasts play a pivotal role during the bioresponse of bone to agents that stimulate bone resorption and/or inhibit bone formation including hormones, polypeptide growth factors, and cytokines. We examined the cytokines interleukin-1-beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alpha) for their effects on osteoblastic proliferation and development and expression of alkaline phosphate and the osteoblast-specific protein osteocalcin in a mineralizing environment. Primary rat osteoblast-like cells (ROB) and osteoblastic cell lines derived from rat (ROS 17/2.8) and human (MG-63) osteosarcomas were studied. IL-1 beta and TNF-alpha were chosen because of their critical importance during the host response to local inflammatory stimuli. Qualitatively similar two- to threefold inhibition of osteocalcin synthesis by IL-1 beta and TNF-alpha were observed in all three postconfluent bone-forming model systems. Because of the readily measurable concentrations of osteocalcin produced in our culture protocol, it was not necessary to enhance osteoblastic synthesis of osteocalcin by supplementation with 1,25(OH)2-vitamin D3, a treatment which exerts pleiotropic effects on osteoblasts. Under the constraints of our protocol, where alkaline phosphatase and mineralization were already elevated at the 14-day onset of treatment, neither of these phenotypic properties was sensitive to a three-day cytokine exposure. Differences were noted in proliferation, where only TNF-alpha stimulated DNA synthesis in ROB cells, while both cytokines stimulated MG-63 cells. IL-1 beta and TNF-alpha failed to alter ROS 17/2.8 DNA synthesis except at the highest doses (25 pM IL-1 beta and 1 nM TNF-alpha) where inhibition was observed. These results further support the view that cytokine-mediated osteoblastic regulation can be relatively selective.