The MAP kinase Fus3 regulates many different signal transduction outputs that govern the ability of Saccharomyces cerevisiae haploid cells to mate. Here we characterize Fus3 localization and association with other proteins. By indirect immunofluorescence, Fus3 localizes in punctate spots throughout the cytoplasm and nucleus, with slightly enhanced nuclear localization after pheromone stimulation. This broad distribution is consistent with the critical role Fus3 plays in mating and contrasts that of Kss1, which concentrates in the nucleus and is not required for mating. The majority of Fus3 is soluble and not bound to any one protein; however, a fraction is stably bound to two proteins of approximately 60 and approximately 70 kDa. Based on fractionation and gradient density centrifugation properties, Fus3 exists in a number of complexes, with its activity critically dependent upon association with other proteins. In the presence of alpha factor, nearly all of the active Fus3 localizes in complexes of varying size and specific activity, whereas monomeric Fus3 has little activity. Fus3 has highest specific activity within a 350- to 500-kDa complex previously shown to contain Ste5, Ste11, and Ste7. Ste5 is required for Fus3 to exist in this complex. Upon alpha factor withdrawal, a pool of Fus3 retains activity for more than one cell cycle. Collectively, these results support Ste5's role as a tether and suggest that association of Fus3 in complexes in the presence of pheromone may prevent inactivation in addition to enhancing activation.