Extracellular space volume fraction, tortuosity and nonspecific uptake of tetramethylammonium--three diffusion parameters of brain tissue--were measured in gray matter of the somatosensory neocortex and subcortical white matter of the rat during postnatal development. The three parameters were determined from concentration-time profiles of tetramethylammonium in postnatal days 2-120 in vivo. Tetramethylammonium concentration was measured with ion-selective microelectrodes positioned 130-200 microns from an iontophoretic source. Data were correlated with cytoarchitectonic structure and average thickness of the regions in 0-90-day-old rats using rapidly frozen tissue. Extracellular space volume fraction was largest in the newborn rats and diminished with age. In two-to three-day-old animals, volume fraction (mean +/- S.E.) was 0.36 +/- 0.04 in layers III and IV, 0.38 +/- 0.02 in layer V, 0.41 +/- 0.01 in layer VI and 0.46 +/- 0.01 in white matter. The earliest decrease in volume fraction was found in layers V and VI at postnatal days 6-7 followed by a decrease in layer III and IV at postnatal days 8-9 and in white matter at postnatal days 10-11. A further dramatic reduction in volume fraction occurred in all cortical layers and especially in the white matter between postnatal days 10 and 21. There was no further decrease in volume fraction between postnatal day 21 and adults (90-120 days old). The adult volume fraction values were: layer II, 0.19 +/- 0.002; III, 0.20 +/- 0.004; IV, 0.21 +/- 0.003; V, 0.22 +/- 0.003; VI, 0.23 +/- 0.007; white matter, 0.20 +/- 0.008. Values of tortuosity ranged between 1.51 and 1.65, nonspecific cellular uptake varied from 3.3 x 10(-3)/s to 6.3 x 10(-3)/s. The variations in each parameter were not statistically significant at any age. These data represent the first characterization of diffusion parameters in a developing brain. They confirm previous histological indications of a relatively large extracellular volume fraction during early postnatal development. The constancy of the tortuosity shows that diffusion of small molecules is no more hindered in the developing brain than in the adult. The large extracellular space volume fraction of the neonatal brain could significantly dilute ions, metabolites and neuroactive substances released from cells, relative to release in adults, and may be a factor in preventing anoxia, seizure and spreading depression in young animals. The diffusion characteristics could also play an important role in the developmental process itself.