Wsc proteins have been identified in fungi and are believed to be stress sensors in the cell wall integrity (CWI) signaling pathway. In this study, we characterized the sensor orthologs WscA and WscB in Aspergillus nidulans. Using hemagglutinin-tagged WscA and WscB, we showed both Wsc proteins to be N-and Oglycosylated and localized in the cell wall and membrane, implying that they are potential cell surface sensors. The wscA disruptant (⌬wscA) strain was characterized by reduced colony and conidia formation and a high frequency of swollen hyphae under hypo-osmotic conditions. The deficient phenotype of the ⌬wscA strain was facilitated by acidification, but not by alkalization or antifungal agents. In contrast, osmotic stabilization restored the normal phenotype in the ⌬wscA strain. A similar inhibition was observed in the wscB disruptant strain, but to a lesser extent. In addition, a double wscA and wscB disruptant (⌬wscA ⌬wscB) strain was viable, but its growth was inhibited to a greater degree, indicating that the functions of the products of these genes are redundant. Transcription of ␣-1,3-glucan synthase genes (agsA and agsB) was significantly altered in the wscA disruptant strain, resulting in an increase in the amount of alkali-soluble cell wall glucan compared to that in the wild-type (wt) strain. An increase in mitogen-activated protein kinase (MpkA) phosphorylation was observed as a result of wsc disruption. Moreover, the transient transcriptional upregulation of the agsB gene via MpkA signaling was observed in the ⌬wscA ⌬wscB strain to the same degree as in the wt strain. These results indicate that A. nidulans Wsc proteins have a different sensing spectrum and downstream signaling pathway than those in the yeast Saccharomyces cerevisiae and that they play an important role in CWI under hypo-osmotic and acidic pH conditions.