Inhibition of gap-junctional intercellular communication (GJIC) via exposure to various toxic substances has been implicated in tumor promotion. In the present study, cigarette smoke total particulate matter (TPM), a known inhibitor of GJIC, were used to characterize a new GJIC screening assay in three independent experiments. The main features of this assay were automated fluorescence microscopy combined with non-invasive parachute technique. Rat liver epithelial cells (WB-F344) were stained with the fluorescent dye Calcein AM (acetoxymethyl) and exposed to TPM from the Kentucky Reference Cigarette 2R4F (a blend of Bright and Burley tobaccos) and from two single-tobacco cigarettes (Bright and Burley) for 3 h. Phorbol-12-myristate-13-acetate (TPA) was used as positive control and 0.5% dimethyl sulfoxide (DMSO) as solvent control. The transfer of dye to adjacent cells (percentage of stained cells) was used as a measure of cellular communication. A clear and reproducible dose–response of GJIC inhibition following TPM exposure was seen. Reproducibility and repeatability measurements for the 2R4F cigarette were 3.7% and 6.9%, respectively. The half-maximal effective concentration values were 0.34 ng/ml for TPA, 0.050 mg/ml for the 2R4F, 0.044 mg/ml for the Bright cigarette, and 0.060 mg/ml for the Burley cigarette. The assay was able to discriminate between the two single-tobacco cigarettes (P< 0.0001), and between the single-tobacco cigarettes and the 2R4F (P = 0.0008, 2R4F vs. Burley and P< 0.0001, 2R4F vs. Bright). Thus, this assay can be used to determine the activity of complex mixtures such as cigarette smoke with high throughput and high precision.