Acetyl-CoA carboxylase (ACC) catalyzes the first and, possibly, the rate-limiting step in fatty acid biosynthesis. Because oligodendrocytes must synthesize large amounts of lipid during myelination, the hypothesis was proposed that ACC might be localized in cells of that type. In sections from the brains of 12-day-old rats, ACC immunostaining was observed in glial cells in white matter and gray matter. These cells resembled carbonic anhydrase-positive oligodendrocytes at mature and immature stages of their development. Cells resembling typical oligodendrocytes were also ACC-positive in white matter from the forebrains and brainstems of 15-17 day-old-rats. In both the gray matter and the white matter of 21-day-old rats there were intensely ACC-positive cells that strongly resembled oligodendrocytes. Oligodendrocytes in the brains of adult rats also were ACC-positive. While recognizing that some ACC must be present at lower levels in other types of cells and at all ages, it was concluded that the present findings are consistent with its primary locus as the oligodendrocytes, particularly during myelination. Further, enrichment of ACC and carbonic anhydrase in the same type of cell suggested that carbonic anhydrase might serve in providing a substrate, bicarbonate, to be utilized by ACC.