The Na+ channel activity (tetrodotoxin sensitive 22Na+ flux induced by veratridine and/or anemone toxin II) was studied in two fractions of brain cell plasma membranes, named A and B, isolated by the method of Gray and Whittaker ((1962) J. Anat. 96, 79-87) from rats 5, 10, 30 and 60 days old. The 22Na+ flux was measured in membrane vesicles formed by the isolated membranes, in the absence of drugs (control), in the presence of veratridine, and in the presence of veratridine plus tetrodotoxin. Fraction A consists primarily of neuronal and glial membranes in rats of 5 and 10 days of age, while in the older rats this fraction becomes enriched in myelin. In Fraction A of 5-day-old and 10-day-old rats, veratridine (25 microM) increases the 22Na+ flux 2.4- and 1.6-fold, respectively, and the increment continues to diminish with age, until it becomes negligible in the 60-day-old rats. Fraction B consists of synaptosomes and membrane vesicles, and at the four ages studied veratridine (25 microM) causes an increment of the 22Na+ flux of about 2.5-fold. Fractions A and B from 10-day-old rats, and Fraction B from 60-day-old rats, which are sensitive to veratridine, also respond to anemone toxin II. When veratridine is used in presence of anemone toxin II (0.5 microM), the K0.5 for veratridine is diminished and the maximum 22Na+ flux is increased. The increments of 22Na+ flux caused by veratridine and/or anemone toxin II in Fractions A and B are blocked by tetrodotoxin (K0.5 approx. 5 nM). Fraction A from 60-day-old rats could be subfractionated by osmotic shock and sucrose gradient centrifugation to obtain three subfractions, two of which are enriched in axolemma and display Na+ channel activity. The other subfraction is enriched in myelin and shows no Na+ channel activity. The plasma membrane preparations from young rats (up to 10 days) are devoid of myelin and are useful for studies of Na+ channel activity.