Adenylate cyclase (AC) toxin from Bordetella pertussis intoxicates eukaryotic cells by increasing intracellular cyclic AMP (cAMP) levels. In addition, insertion of AC toxin into the plasma membrane causes efflux of intracellular K⁺ and, in a related process, hemolysis of sheep erythrocytes. Although intoxication, K⁺ efflux, and hemolysis have been thoroughly investigated, there is little information on the nature of the interaction of this toxin with intact target cells. Using flow cytometry, we observe that binding of AC toxin to sheep erythrocytes and Jurkat T lymphocytes is dependent on posttranslational acylation of the toxin. Extracellular calcium is also necessary, with a steep calcium concentration dependence similar to that required for intoxication and hemolysis. Binding of AC toxin is concentration dependent but unsaturable up to 50 µg/ml, suggesting that if there is a specific receptor molecule with which the toxin interacts, it is not limiting. Visualization of cells by fluorescence microscopy supports the data obtained by flow cytometry and reveals a peripheral pattern of toxin distribution. AC toxin binds to erythrocytes at both 0 and 37°C; however, the total binding at 0°C is less than that at 37°C. In human erythrocytes, AC toxin does not cause an increase in K⁺ efflux or hemolysis. While AC toxin exhibits reduced potency to increase cAMP in these cells than in sheep erythrocytes, there is only a modest reduction in the binding of the toxin as measured by flow cytometry. Further use of this technique will provide new approaches for dynamic and functional analysis of the early steps involved in intoxication, K⁺ efflux, and hemolysis produced by AC toxin.