A primary culture system of equine uterine epithelial cells was developed to study interactions between these cells in vitro. Cultures were prepared by combined mechanical and collagenase separation of cells from endometrial biopsies taken from mares during estrus and diestrus. Cells were maintained in DME/F12 medium containing 10% fetal bovine serum. The purity of the epithelial cell population was estimated to be greater than 95% based upon the cobblestone morphology of cells and a uniform indirect immunofluorescence labelling of cells with an antibody directed against cytokeratin intermediate filament proteins. Radiating cytoplasmic cytokeratin filaments were characteristic of cells labelled after 3 days in culture. Preliminary analysis of the incidence and rate of cell-to-cell communication between epithelial cell populations was conducted using a fluorescence recovery after photobleaching assay. Recovery of carboxyfluorescein fluorescence in cells which were laser photobleached and in contact with other cells was monitored at one-minute intervals over 5 minutes. The incidence of communication among cells isolated during estrus and diestrus was 100%. The mean rates of fluorescence recovery at one minute (8.25% ± SEM 0.91 and 10.94% ± SEM 1.20 for cells obtained from estrous and diestrous mares, respectively) were not significantly different. These studies indicate that cells isolated from the endometrium of the mare are capable of intercellular diffusion of regulatory and informational molecules between cells in contact.