The biology of Candida albicans, including dimorphism and virulence, is significantly influenced by environmental pH. The response to ambient pH includes the pH-conditional expression of several genes, which is directly or indirectly regulated by Rim101p. Rim101p is homologous to PacC, a transcription factor that regulates pH-conditional gene expression in Aspergillus nidulans. PacC binds 5'-GCCARG-3' sequences upstream of pH-responsive genes and either activates or represses transcription. The absence of pacC consensus binding sites upstream of PHR1, a RIM101-dependent, alkaline pH-induced gene of C. albicans, suggested either that PHR1 is indirectly regulated by Rim101p or that the binding specificity of Rim101p is different. In vitro binding studies demonstrated that Rim101p strongly bound two regions upstream of PHR1 that were only weakly bound by PacC. Deletion analysis and site-specific mutagenesis demonstrated that both sites were functionally significant, mutation of either site reduced RIM101-dependent induction, and expression was abolished in the double mutant. Furthermore, oligonucleotides containing these sites conferred pH-conditional expression when inserted upstream of a reporter gene. The consensus sequence of these sites, 5'-CCAAGAAA-3', was identical to the binding recognition sequence identified by in vitro selection of Rim101p binding oligonucleotides from a random pool. The functional significance of this binding sequence was reinforced by its observed presence upstream of a number of newly identified pH-conditional genes. We conclude that Rim101p acts as a transcription factor and directly regulates pH-conditional gene expression but has a binding specificity different from that of PacC.