Abstract: BACKGROUND: Signaling through mitogen-activated protein kinase (MAPK) cascade pathways can show various input-output behaviors, including either switch-like or graded responses to increasing levels of stimulus. Prior studies suggest that switch-like behavior is promoted by positive feedback loops and nonprocessive phosphorylation reactions, but it is unclear whether graded signaling is a default behavior or whether it must be enforced by separate mechanisms. It has been hypothesized that scaffold proteins promote graded behavior. RESULTS: Here, we experimentally probe the determinants of graded signaling in the yeast mating MAPK pathway. We find that graded behavior is robust in that it resists perturbation by loss of several negative-feedback regulators. However, the pathway becomes switch-like when activated by a crosstalk stimulus that bypasses multiple upstream components. To dissect the contributing factors, we developed a method for gradually varying the signal input at different pathway steps in vivo. Input at the beginning of the kinase cascade produced a sharp, threshold-like response. Surprisingly, the scaffold protein Ste5 increased this threshold behavior when limited to the cytosol. However, signaling remained graded whenever Ste5 was allowed to function at the plasma membrane. CONCLUSIONS: The results suggest that the MAPK cascade module is inherently ultrasensitive but is converted to a graded system by the pathway-specific activation mechanism. Scaffold-mediated assembly of signaling complexes at the plasma membrane allows faithful propagation of weak signals, which consequently reduces pathway ultrasensitivity. These properties help shape the input-output properties of the system to fit the physiological context.