The cholesterol content of normal human erythrocyte membranes (ghosts) was reduced by incubation of the ghosts with phosphatidylcholine (PC) liposomes for 4 h at 37 °C. As controls, ghosts were stored at 4 °C, incubated alone or incubated with PC liposomes saturated with cholesterol; none of these treatments altered membrane cholesterol content. The activities of three membrane-bound enzymes, spectrin kinase, band-3 protein kinase, and phospholipid kinase were reduced in cholesterol-depleted ghosts compared with control ghosts (p < 0.02, p < 0.05, and p < 0.01, respectively). Reductions of the protein kinase activities ranged from 5 to 30% and were apparently related to the extent of the reduction of ghost cholesterol content. The reduction of phospholipid kinase activity was greater (30–60%) and showed no correlation with the extent of reduction of ghost cholesterol content. In contrast to the kinase activities, the activity of a fourth enzyme, NADH: cytochrome c oxidoreductase, increased in response to membrane cholesterol depletion. The relationship between the increase in oxidoreductase activity and cholesterol depletion was best described as linear (r = 0.76).The data demonstrate that these enzyme activities are affected by membrane cholesterol content. Since the alterations in the enzyme activities caused by the artificial alterations of membrane cholesterol content were comparable with those observed in vivo in various pathological conditions, we suggest that the observed changes may be due to mechanisms whereby membrane lipids influence membrane enzyme activities in vivo.