In order to gain insight into the physical interaction between bacterial endotoxins and the surface of human monocytes, we investigated the effects of Salmonella typhi endotoxin and lipid A on two functional properties of the plasma membrane of these cells: (1) the transmembrane electrical potential and (2) the fluidity of the lipid bilayer. Using the fluorescent lipophilic cationic probe 3,3'-dipropylthiodicarbocyanine (di-S-C3(5] to monitor the transmembrane electrical potential, we found that neither endotoxin nor lipid A induced depolarization of the monocyte's plasma membrane or impeded its ability to undergo depolarization in response to phorbol myristate acetate. When the resting transmembrane potential of the monocyte was analyzed by exposing di-S-C3(5)-labeled cells suspended in media containing incremental concentrations of potassium ion (K+) to valinomycin, no difference between the response of control cells and cells pretreated with endotoxin was noted. We next examined the effect of endotoxin and lipid A on the fluidity of the monocyte's plasma membrane by monitoring the intensity of the fluorescence of 1,6-diphenyl-1,3,5-hexatriene. By quantifying the intensity of parallel and perpendicular polarized light emitted by this membrane-embedded probe between 8 and 56 degrees C, measurements of molecular anisotropy were used to identify temperature-dependent phase transitions within the hydrocarbon region of the plasma membrane and to estimate the relative microviscosity of the lipid bilayer before and after exposing the cells to endotoxin or lipid A. Although the temperature at which phase transitions occurred was the same in all experimental groups of cells, preincubation of monocytes with either endotoxin or lipid A appeared to increase both the apparent microviscosity of the cell membrane and the order of the lipid bilayer as reflected by a decrease in its flow-activation energy. Our data indicate that when endotoxin molecules contact the surface of the monocyte, the lipid A moiety appears to become incorporated into the plasma membrane, increasing the microviscosity of the lipid bilayer without significantly altering its ionic permeability. We therefore conclude that the metabolic activation of monocytes by endotoxin is not coupled to, or initiated by, membrane depolarization.