Abstract: Several biochemical parameters were analyzed in cultured embryonic mouse spinal cord during various stages of normal myelinogenesis or demyelination. In cultures demyelinated by exposure to anti‐whole CNS tissue serum plus complement, the activity of 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase (EC 3.1.4.37) was decreased 70%, whereas in cultures that did not show morphological changes with complement‐inactivated anti‐CNS serum or anti‐myelin basic protein serum, the activity was 30% lower than in control cultures. The lipid composition of these cultures was quantitated by means of high‐performance thin‐layer chromatography densitometry technique. Cultures with normal nutrient medium alone or with the addition of 5% normal rabbit serum plus 10% guinea pig serum had 30% of the total lipid content of that present in newborn mouse spinal cord of the corresponding age. There were, however, relatively more lysophospholipids, cholesterol esters, triglycerides, and free fatty acids and less phosphatidylethanolamine and galactolipids in cultures as compared with normal spinal cord. Explants demyelinated by exposure to anti‐CNS serum plus complement demonstrated principally a 70% decrease in the content of galactolipids with respect to normal cultures. When complement was inactivated, total lipids increased 42% (with increases of 40–70% in individual lipids). Inclusion of anti‐myelin basic protein serum plus complement in the medium produced no significant changes in the lipid composition of the cultures.