The incorporation of the fluorescent sterol analog, cholesta-5,7,9-trien-3β-ol, into rat brain P2 fractions and its feasibility to detect membrane perturbations by polarization analysis were evaluated. This investigation involved the administration (i.c.v.) of the fluorescent compound in sesame oil with subsequent determination of optimal dose and the half-life of this compound within the membrane fraction. The dose giving maximum polarization was found to be 0.65 μmole, and pharmacokinetic analysis revealed that the disappearance of this analog followed a two-compartment open model with an elimination half-life from the membrane fraction of 655 hr. Increasing temperature of the membrane preparation showed an increase in polarization, indicating a consequent decrease in mobility of the molecule, while addition of a fluidizing agent like ethanol caused a decrease in polarization, and thus the expected increase in fluidity.