In the present work, several preparatory procedures commonly used for electron microscopy (EM) were evaluated as to their ability to preserve cholesterol (CHO) and CHO derivatives in tissue. We also determined in several rat tissues to what extent the sterols used as tracers are metabolized. Sprague-Dawley rats were injected intraperitoneally with [1 alpha,2 alpha(n)-3H]cholesterol ([3H]CHO) and 25-hydroxy-[26,27-3H]cholesterol ([3H]25-OH-CHO). Lipids of the liver, aorta and brain were extracted one and five days after injection, and the distribution of the labeled lipids was followed by thin-layer chromatography. When labeled CHO was injected as tracer, most of the radioactivity remained associated with the CHO fraction. When 25-hydroxycholesterol (25-OH-CHO) was used, we found that it was mostly metabolized to yield more polar compounds. Our results show that the loss of CHO and CHO derivatives from tissues depends not only on the preparatory procedure used for EM, but also on the type of tissue studied.