NC1175 (3-[3-(4-chlorophenyl)-2-propenoyl]-4-[2-(4-chlorophenyl)vinylene]-1-ethyl-4-piperidinol hydrochloride) is a novel thiol-blocking conjugated styryl ketone that exhibits activity against a wide spectrum of pathogenic fungi. Incubation of NC1175 with various concentrations of cysteine and glutathione eliminated its antifungal activity in a concentration-dependent fashion. Since NC1175 is a lipophilic compound that has the potential to interact with cytoplasmic membrane components, we examined its effect on the membrane-located proton-translocating ATPase (H⁺-ATPase) of yeast (Candida albicans, Candida krusei, Candida guilliermondii, Candida glabrata, and Saccharomyces cerevisiae) and Aspergillus (Aspergillus fumigatus, Aspergillus niger, Aspergillus flavus, and Aspergillus nidulans) species. The glucose-induced acidification of external medium due to H⁺-ATPase-mediated expulsion of intracellular protons by these fungi was measured in the presence of several concentrations of the drug. NC1175 (12.5 to 50 µM) inhibited acidification of external medium by Candida, Saccharomyces, and Aspergillus species in a concentration-dependent manner. Vanadate-inhibited hydrolysis of ATP by membrane fractions of C. albicans was completely inhibited by 50 µM NC1175, suggesting that the target of action of NC1175 in these fungi may include H⁺-ATPase.