In the yeast plasma-membrane H(+)-ATPase and other P-type ATPases, conformational changes are transmitted between cytoplasmic and membrane-embedded domains via a stalk region composed of cytoplasmic extensions of membrane segments 2, 3, 4, and 5. The present study has used a fluorescent maleimide (Alexa-488) to probe Cys residues introduced into stalk segments 4 and 5 of the yeast enzyme. In the case of S5, Cys substitutions along one face led to a constitutive, 5- to 10-fold activation of the ATPase in the absence of glucose. Based on homology with SERCA Ca(2+)-ATPase, this face is likely to be buried in the interior of the protein, close to the P domain. Three Cys residues on the opposite face of S5 (A668C, S672C, and D676C) were accessible to Alexa-488 under all conditions tested. In addition, three other Cys residues at or near the boundary between the two faces reacted with Alexa-488 only (V665C, L678C) or preferentially (Y689C) in plasma membranes from glucose-metabolizing cells; this result provides the first direct evidence for a change in conformation of S5 during glucose activation. For stalk segment 4, site-directed mutagenesis gave no sign of a role in glucose-dependent regulation. Rather, substitutions at 13 consecutive positions along S4 caused kinetic changes consistent with a shift in equilibrium from E2 to E1. Four Cys residues along this stretch of S4 (Q357C, K362C, S364C, and S368C) reacted with Alexa-488, indicating that they are exposed to the aqueous medium as predicted in the SERCA-based structural model.