Analyses of intercellular communication is useful for assessing the effects of chemical treatment on the function of mammalian cell membranes in vitro. The objective of this study was to quantify and compare the activity of mainstream cigarette smoke condensate (CSC) from tobacco-heating and tobacco-burning cigarettes on both the rate and total amount of intercellular communication in vitro. Lucifer yellow uptake and lactate dehydrogenase release assays were used to evaluate plasma membrane toxicity. Gap junction intercellular communication (GJIC) was determined by quantifying fluorescence redistribution after photobleaching (FRAP) following a 1-hr exposure to concentrations of CSCs which were not toxic to the plasma membrane. GJIC was quantified in rat hepatic epithelial cells (WB cells) and human skin fibroblasts (MSU-2 cells) synchronized in the G1 phase of the cell cycle. In each of the cell types tested, CSC from tobacco-heating cigarettes did not inhibit GJIC at concentrations, where CSC from tobacco-burning cigarettes significantly inhibited both the total amount and the rate of GJIC. These results indicate that mainstream smoke condensate of cigarettes which heat tobacco is less biologically active than mainstream smoke condensate of cigarettes that burn tobacco as determined by in vitro gap junction intercellular communication.