Different environmental stimuli often use the same set of signaling proteins to achieve very different physiological outcomes. The invasive growth and mating pathways in yeast Saccharomyces cerevisiae each employ a mitogen-activated protein (MAP) kinase cascade that includes several common components (e.g. Ste11, Ste7), after which the pathways diverge leading to activation of different MAP kinases (Fus3 and Kss1, respectively). In order to determine how pathway specificity is achieved, we conducted a quantitative analysis of MAP kinase activity, and devised a series of six computational models that describe their behavior in vivo. As anticipated by one of the models, MAP kinase phosphorylation of shared pathway components results in diminished activation of one MAP kinase but not the other. These findings reveal how one MAP kinase can limit the activity of a competing MAP kinase and thereby ensure signal fidelity. Supported by National Institutes of Health GM073180 (H. G.) and GM073180 (T. E.) and the American Heart Association 0415413U (N. H.).