Experiments using confocal laser microscopy on the rat osteosarcoma cell line (ROS 17/2.8) indicate that mechanical stimulation elicits pronounced [Ca2+](i)transients in the MS (mechanically stimulated) cell, which then propagate to the NB (neighbouring) cells. Experiments with Ca(2+)-free solutions or gadolinium suggest that Ca(2+)-influx through stretch-sensitive channels is required. When intracellular stores are depleted with thapsigargin, mechanical stimulation was able to evoke a Ca(2+)transient of reduced amplitude that disappeared entirely after subsequent blocking of Ca(2+)-influx. Heptanol inhibited intercellular propagation of the Ca(2+)transient, demonstrating the involvement of gap junctions in the propagation of the Ca(2+)transient in ROS cells. PKC activation has only a small inhibitory effect, while inhibition of PKC or tyrosine kinase was ineffective. PKA activation reduced the amplitude of the [Ca2+](i)-rise in NB cells, and decreased the percentage of responsive cells. Cells grown in 50mM glucose for 72h presented only a very limited decrease of the Ca(2+)-rise during mechanical stimulation in the MS and NB cells compared to control conditions. PKC downregulation in high glucose did not modulate this effect.The results of our experiments indicate that PKC or sustained high glucose concentrations do not affect gap junctional communication in ROS cells, while activation of PKA has an inhibitory effect. This might indicate that osteoblastic dysfunction in diabetes could be directly related to the high glucose concentrations and not to inhibition of the intercellular communication.