The effect of sodium butyrate on membrane phospholipid metabolism in a neonate rat cerebellum derived clonal oligodendrocyte cell line (CB-II) was investigated. Sodium butyrate is an agent known to induce cell differentiation and morphological transformations. A comparison of the in vivo phospholipid labeling patterns obtained by incubating CB-II cells with [3H]choline, [14C]myristic acid or [3H]arachidonic acid indicated that butyrate altered the route of acylation-deacylation in phosphatidylcholine (PC) biosynthesis. Using an in vitro incubation system containing homogenates of CB-II cells, the largest proportion of radioactivity was found in PC, and addition of sodium butyrate resulted in a further increase in the transfer of arachidonic acid to PC, but not to phosphatidylinositol. Similar results were obtained when this in vitro acylation activity was tested using homogenates from sodium butyrate pretreated cells. The butyrate effect was observed regardless of whether or not exogenous lysophosphatidylcholine (LPC) was added to the incubation system. Addition of butyrate did not result in a change in the activity of LPC:acyl-CoA (coenzyme A) acyltransferase (EC 2.3.1.23) in CB-II cells upon incubating cell homogenates with [1-14C]arachidonoyl-CoA and LPC. However, when cell homogenates were incubated with [3H]arachidonic acid in the presence of 2.5-10 mM sodium butyrate, arachidonoyl-CoA synthesis was stimulated. A time course study demonstrated that significant stimulation occurred after three minutes. Taken together, the results suggest that in CB-II cells, sodium butyrate stimulates the transfer of arachidonic acid into PC and that this effect is at least partially due to a stimulation of arachidonoyl-CoA ligase (EC 6.2.1.3).