Direct cytotoxic effects of radiocontrast (RC) agents have been implicated in radiocontrast nephropathy (RCIN). The interaction between extracellular calcium, which plays a central role in intercellular contacts, and the in vitro toxicity of RC was tested in Madin-Darby canine kidney (MDCK) cell monolayers grown on permeable supports. Cell viability was determined by trypan blue exclusion. The function of intercellular junctions was assessed by measuring the electrical transmonolayer resistance (TMR). The cell contacts were examined with indirect immunofluorescence microscopy using antibodies against the junctional proteins E-cadherin, ZO-1 and occludin. The ionic RC agents diatrizoate and ioxaglate (74 mg iodine/ml), but not the nonionic compounds iohexol or iodixanol, decreased ionized calcium (Ca<sup>2+</sup>) in the incubation media from 1.48 ± 0.04 m M (control) to 0.89 ± 0.06 m M (diatrizoate), respectively to 1.05 ± 0.08 m M (ioxaglate). Diatrizoate, and to a lesser extent ioxaglate, reduced the number of viable MDCK cells and showed a redistribution of the E-cadherin, ZO-1 and occludin immunofluorescence signal with a parallel decrease of the TMR indicating an impaired monolayer integrity. A similar reduction of extracellular Ca<sup>2+</sup> through EGTA failed to reproduce these effects. Conversely, raising Ca<sup>2+</sup> in diatrizoate-containing media to control levels did not abrogate its toxicity. In conclusion, the ionic RC agents diatrizoate and ioxaglate, but not the nonionic compounds iohexol or iodixanol, reduce extracellular Ca<sup>2+</sup> in vitro. However, this reduction of Ca<sup>2+</sup> does not explain their cytotoxic effects which could contribute to the pathogenesis of RCIN in vivo by opening intercellular junctions.