Myelinogenic cultures of cells dissociated from embryonic mouse brain were used to study the regulation of myelination-associated molecules by cortisol. Cortisol in physiological concentrations (0.03 microM) caused an increased accumulation of myelination-associated sulphogalactolipids. It also stimulated the myelin- and oligodendroglia-specific cyclic nucleotide phosphohydrolase. The increase in sulphogalactolipid content was caused by a cortisol-concentration-dependent inhibition in arylsulphatase A activity and not by an increase in either cerebroside sulphotransferase activity or an increase in availability of adenosine 3'-phosphate 5'-phosphosulphate. Of several steroid hormones tested only the glucocorticoid types brought about these changes. The relationship between net sulphogalactolipid accumulation and arylsulphatase A inhibition induced by cortisol was confirmed by sulphogalactolipid turnover studies. Depending on whether a single-phase or a two-phase decay calculation is used, the turnover of sulphogalactolipid with cortisol present was decreased at 22 days in culture by either 62% or 65% respectively of that without cortisol. This decrease in turnover can be attributed completely to the decrease of arylsulphatase activity by cortisol to 63% of the value for normal cells grown under the same conditions.