Heat shock proteins are molecular chaperones linked to a myriad of physiological functions in both prokaryotes and eukaryotes. In this study, we show that the Aspergillus nidulans hsp30 (ANID_03555.1), hsp70 (ANID_05129.1), and hsp90 (ANID_08269.1) genes are preferentially expressed in an acidic milieu, whose expression is dependent on the palA + background under optimal temperature for fungal growth. Heat shock induction of these three hsp genes showed different patterns in response to extracellular pH changes in the palA + background. However, their accumulation upon heating for 2 h was almost unaffected by ambient pH changes in the palA − background. The PalA protein is a member of a conserved signaling cascade that is involved in the pH-mediated regulation of gene expression. Moreover, we identified several genes whose expression at pH 5.0 is also dependent on the palA + background. These results reveal novel aspects of the heat-and pH-sensing networks of A. nidulans.