The present study investigated the effects of elevated cytoplasmic free calcium concentrations ([Ca2+]i) on the permeability of gap junctions between cultured osteoblast-like (OB) cells derived from calvarial and periosteal fragments of newborn rats. This was studied using the double whole cell patch clamp technique and intracellular dye injections. To increase [Ca2+]i, patch pipette solutions contained 100 micromol/liter Ca2+. About 1-2 minutes after whole cell modes had been attained, the total number of gap junction channels was reduced from an average of 400 in normal Ca2+ to 20 in high Ca2+. Thereafter, remaining gap junction channels were active for up to 8 minutes. In normal rat kidney (NRK) cells, gap junction channels were closed by high Ca2+ within 1 minute, pointing to a similar sensitivity of Connexin43 gap junction channels in OB and NRK cells. To study the effects of elevated [Ca2+]i on the dye permeability of gap junctions between extended OB cells, the spread of Lucifer Yellow to neighboring cells was evaluated. [Ca2+]i was gradually increased from 1.5- to 14-fold the normal value by application of either ouabain, Na+-free/ouabain, or A23187. Reduced dye spread correlated with the increase of [Ca2+]i measured by analyzing the fluorescence of fura-2. These data show that gap junctions in OB cells are sensitive to Ca2+.