Biological membranes are supposed to have heterogeneous structure containing lipid rafts-lateral micro- and nanodomains enriched in cholesterol (chol) and sphingolipids. In this work, lipid bilayers containing a small amount of the spin-labeled chol analogue 3β-doxyl-5α-cholestane (chlstn) were studied using electron spin echo (ESE) spectroscopy, which is a pulsed version of electron paramagnetic resonance (EPR). Bilayers were prepared from an equimolecular mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with chol added at different concentrations. The ESE decays recorded at 77 K become faster with increase of chlstn concentration. The chlstn-dependent contribution to ESE decay is remarkably nonexponential; however, the logarithm of this contribution can be rescaled for different chlstn concentrations to a universal function with the rescaling factor approximately proportional to concentration. This result shows that the chlstn-dependent contribution to the ESE decay can be employed to estimate the local (at the nanometer scale of distances) chlstn concentration. Analogous rescaling behavior is also observed for the bilayers with different chol concentrations, with the rescaling factor increasing with increase of the chol concentration. This result is evidence that chlstn molecules are distributed heterogeneously in the chol-containing bilayer and form clusters with enhanced chlstn (and probably chol) local concentration. The local concentration of chlstn molecules for large chol content (∼30 mol %) was enhanced by at least ∼70% versus chol-free bilayers. The suggested approach appears to be useful for exploring heterogeneities in lipid composition of biological membranes of different types.