A whole-cell model of a macrophage (mphi) is developed to simulate pH and volume regulation during a NH4Cl prepulse challenge. The cell is assumed spherical, with a plasma membrane that separates the cytosolic and extracellular bathing media. The membrane contains background currents for Na+, K+ and Cl-, a Na(+)-K+ pump, a V-type H(+)-extruder (V-ATPase), and a leak pathway for NH4+. Cell volume is controlled by instantaneous osmotic balance between cytosolic and extracellular osmolytes. Simulations reveal that the mphi model can mimic alterations in measured pH(i) and cell volume (Vol(i)) data during and after delivery of an ammonia prepulse, which induces an acid load within the cell. Our analysis indicates that there are substantial problems in quantifying transporter-mediated H+ efflux solely from experimental observations of pH(i) recovery, as is commonly done in practice. Problems stemming from the separation of effects arise, since there is residual NH4+ dissociation to H+ inside the mphi during pH(i) recovery, as well as, proton extrusion via the V-ATPase. The core assumption of conventional measurement techniques used to estimate the H+ extrusion current (I(H)) is that the recovery phase is solely dependent on transporter-mediated H+ extrusion. However, our model predictions suggest that there are major problems in using this approach, due to the complex interactions between I(H), NH3/NH4+ buffering and NH3/NH4+ efflux during the active acid extrusion phase. That is, the conventional buffer capacity-based I(H) estimation must also take into account the perturbation that a prepulse challenge brings to the cytoplasmic acid buffer itself. The importance of this whole-cell model of mphipH(i) and volume regulation lies in its potential for extension to the characterization of several other types of non-excitable cells, such as the microglia (brain macrophage) and the T-lymphocyte.