Osteoblasts and adipocytes are thought to differentiate from a common stromal progenitor cell. These two phenotypically mature cell types show a high degree of plasticity, which can be observed when cells are grown under specific culture conditions. Gap junctions are abundant among osteoblastic cells in vivo and in vitro, whereas they are down-regulated during adipogenesis. Gap junctional communication (GJC) modulates the expression of genes associated with the mature osteoblastic phenotype. Inhibition of GJC utilizing 18-alpha-glycyrrhetinic acid (AGRA) blocks the maturation of pre-osteoblastic cells in vitro. Moreover, cytoplasmic lipid droplets are detectable at the end of the culture period, suggesting that GJC inhibition may favor an adipocytic phenotype. We used several human osteoblastic cell lines, as well as bone-derived primary osteoblastic cells, to show that confluent cultures of human osteoblastic cells grown under osteogenic conditions developed an adipocytic phenotype after 3 days of complete inhibition of GJC using AGRA or oleamide, two dissimilar nontoxic reversible inhibitors. Development of an adipogenic phenotype was confirmed by the accumulation of triglyceride droplets and the increase in mRNA expression of the adipocytic markers peroxisome proliferator-activated receptor gamma2 and lipoprotein lipase. Glycyrrhizic acid, a noninhibitory AGRA analog, or alpha-bromopalmitate, a nondegradable fatty acid, had no effect. Modulation of skeletal GJC may represent a new pharmacological target by which inhibition of marrow adipogenesis can take place with the parallel enhancement of osteoblastogenesis, thus providing a novel therapeutic approach to the treatment of human age-related osteopenic diseases and postmenopausal osteoporosis.