Regulated ubiquitination and degradation of signaling proteins have emerged as a key mechanism for modulating the strength and duration of signaling pathways. The reversible nature of the ubiquitination process as well as the large number and diversity of the deubiquitinating enzymes raise the possibility that signaling pathways might be modulated by specific deubiquitinating enzyme(s). Here we provide evidence that in the yeast Saccharomyces cerevisiae, the Pkc1-mediated signaling pathway that controls the cell wall integrity is negatively regulated by a deubiquitinating enzyme Ubp3. Disruption of UBP3 gene leads to an enhanced activation of the cell wall integrity pathway MAP kinase Slt2 when cells are challenged with a variety of pathway activation agents such as pheromone and Congo red. The ubp3 deletion mutants accumulate high level of Pkc1, suggesting potential regulation of Pkc1 by Ubp3. Consistent with this, Pkc1 and Ubp3 interact in vivo, and the stability of Pkc1 is markedly increased in the ubp3 deletion mutants. Moreover, disruption of PKC1 gene, but not the genes that encode components downstream of Pkc1, completely suppresses other phenotypes displayed by the ubp3 deletion mutants, such as hyper-activation of a pheromone-responsive MAP kinase Fus3 (J. Biol. Chem. 277:15766-72, 2002). These findings demonstrate that Pkc1 can be regulated by Ubp3 that facilitates its destruction, and provide the initial evidence that Pkc1 plays a positive role in modulating the parallel pheromone signaling pathway.