To evaluate whether intercellular coupling via connexin43 gap junction channels modulates hormonal responsiveness of cells in contact, we have created osteoblastic cell lines deficient in connexin43. Osteoblastic ROS 17/2.8 cells were transfected with a plasmid containing an antisense cDNA construct to rat connexin43. Control transfection did not alter cell-to-cell coupling nor connexin43 mRNA or protein expression relative to nontransfected ROS 17/2.8 cells. In contrast, stable transfection with an antisense connexin43 cDNA resulted in two clones, RCx4 and RCx16, which displayed significant decreases in connexin43 mRNA and protein expression and were dramatically deficient in cell-to-cell coupling. Phenotypically, all transfectants retained osteoblastic characteristics. However, cells rendered connexin43-deficient through antisense transfection displayed a dramatic attenuation in the cAMP response to parathyroid hormone. Alterations in hormonal responses were not due to changes in parathyroid hormone receptor number or binding kinetics nor to alterations in adenylyl cyclase activity. These results indicate that gap junctions may be required for mediating hormonal signals. Furthermore, these experiments support a regulatory role for connexin43-mediated intercellular communication in the modulation of hormonal responses within elaborately networked bone cells.