Sequence of reactions in the process of ternary complex formation of BMY-28864 with D-mannopyranoside and calcium was spectrophotometrically determined under more strict analytical conditions using metal-free preparations of sugars and the pradimicin derivative at a bandpass slit width of 1 nm. In the first phase of ternary complex formation, BMY-28864 stereospecifically recognized and bound to D-mannopyranoside in the absence of calcium, which was revealed by a visible absorption maximum shift of ca. 8 nm. Subsequently, the BMY-28864-D-mannopyranoside conjugate reacted with calcium to yield the ternary complex, which was detected by an additional visible absorption maximum shift of ca. 8 nm. When the three components were mixed at the same time, both phases simultaneously occurred to produce the ternary complex which was accompanied by a visible absorption maximum shift of 16 nm in total. Based on this two-phased reaction sequence, the mechanism of ternary complex formation of BMY-28864 with D-mannopyranoside and calcium was reexamined in details. Terminal D-mannopyranoside was confirmed to be essential as BMY-28864-specific sugar receptor by in vitro analysis and animal cell experiments. While calcium, strontium and cadmium behaved similar in the in vitro ternary complex formation, the yeast and animal cell experiments showed that only calcium played a dual role as a base in the ternary complex formation and as an effector in physiological disturbances leading to cell death.