Phosphorylation-dephosphorylation cascade systems represent a major mechanism of cellular regulation. A theoretical analysis of such systems (Stadtman & Chock 1977) revealed that they are endowed with extraordinary regulatory capacities; they may exhibit smooth, flexible responses to changes in single and multiple metabolite levels, signal amplification, and apparent positive cooperativity. To test the theories and equations involved in this analysis, an in vitro phosphorylation-dephosphorylation model system was developed. The system consists of a cyclic-AMP-dependent protein kinase and a phosphoprotein phosphatase, both isolated to near-homogeneity from bovine heart, and a nanopeptide that serves as the interconvertible substrate. Experiments with the model system confirm the predictions about the behaviour of a monocyclic cascade. They also reveal that when the concentration of enzyme-substrate complex is not negligible, cyclic cascades are potentially more sensitive to variations in effector concentrations and can achieve even greater signal amplification than previously predicted.