AbstractPrevious results (Fressinaud, C., Sarliève, L.L., and Labourdette, G. J. J. Cell. Physiol.,141:667–674, 1989b) have shown that cerebroside sulfotransferase (CST; EC 2.8.2.11) is enriched in pure rat oligodendrocyte (OL) cultures and that its activity is increased by factors mitogenic for OL precursors and galactocere‐broside (GC) expressing OL, such as basic fibroblast growth factor (bFGF), platelet‐derived growth factor, and high insulin concentrations. In contrast, transforming growth factor β or low insulin concentrations were found to be ineffective in this culture system. As bFGF mainly enhanced the proliferation of OL precursors (GC negative cells) rather than that of differentiated (GC +) cells, a relationship between OL precursor proliferation and CST increase was suggested. This hypothesis was first tested in 20‐day‐old OL cultures grown in chemically defined medium. The dose‐response curve of [125I] lododeoxyuridine ([125I]dUrd) incorporation toward bFGF was parallel to that of CST specific activity, and maximal stimulation was reached at 5 ng/ml bFGF for both. In contrast, 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNP; EC 3.1.4.37) specific activity decreased after bFGF treatment. To determine if CST increase was linked to the proliferation of OL precursors induced by bFGF, cell proliferation was blocked by cytosine arabinoside (ARA‐C). From 10−8to 10−5M ARA‐C there was a dose‐dependent inhibition of cell proliferation and a decrease in CST specific activity, whereas CNP specific activity was enhanced. When the cells were treated with bFGF and 10−6M ARA‐C together, the proliferation was completely blocked and CST activity decreased by 72% below control values, whereas CNP activity was not significantly decreased. Immunocytochemical studies showed that the number of sulfatide‐expressing cells and the number of cycling cells were increased after bFGF treatment, but that there was no overlapping between these two populations. Taken together these results suggest that CST activity and sulfatide expression appear shortly af