Cellular responses to many external stimuli are mediated by mitogen-activated protein kinases (MAPKs). We investigated whether dynamic intracellular movement contributes to the spatial and temporal characteristics of the responses elicited by a prototypic MAPK, Fus3, in the mating pheromone response pathway in budding yeast (Saccharomyces cerevisiae). Confining Fus3 in the nucleus, via fusion to a histone H2B, reduced MAPK activation and diminished all responses (pheromone-induced gene expression, cell cycle arrest, projection formation, and mating). Elimination of MAPK phosphatases restored more robust outputs for all responses, indicating that nuclear sequestration impedes full MAPK activation but does not abrogate its functional competence. Restricting Fus3 to the plasma membrane, via fusion to a lipid-modified CCaaX motif, led to MAPK hyperactivation yet severely impaired all response outputs. Fus3-CCaaX also caused aberrant cell morphology and a proliferation defect. Unlike similar phenotypes induced by pathway hyperactivation via upstream components, these deleterious effects were independent of the downstream transcription factor Ste12. Thus, appropriate cellular responses require free subcellular MAPK transit to disseminate MAPK activity optimally because preventing dynamic MAPK movement either markedly impaired signal-dependent activation and/or resulted in improper biological outputs.