The apparent diffusion coefficient, D*, was measured in rat cortical slices and compared to the free diffusion coefficient, D, for three negatively charged proteins, lactalbumin (mol. wt = 14,500), ovalbumin (45,000) and bovine serum albumin (66,000). The temporal evolution of the spatial distribution of albumin molecules labeled with the Texas Red fluorophore was determined using integrative optical imaging at intervals after a brief pressure injection from a micropipette in slices of adult rat cerebral cortex and dilute agarose gel. Diffusion coefficients were obtained by fitting appropriate equations to the data. In slices at 34 degrees C, the values of D* (10(-7) cm2/s, mean +/- S.E.M.) for lactalbumin, ovalbumin and bovine serum albumin were 2.37 +/- 0.10, 1.60 +/- 0.08 and 1.63 +/- 0.07, respectively. In agarose gel, values of D (10(-7) cm2/s) were 11.87 +/- 0.20, 10.02 +/- 0.25 and 8.29 +/- 0.17, respectively. From these data the tortuosity factors, (D/D*)0.5, were calculated, with 2.24 obtained for lactalbumin, 2.50 for ovalbumin and 2.26 for bovine serum albumin. Previous optical measurements using dextrans with mol. wts of 40,000 and 70,000 gave tortuosities of 2.16 and 2.25, but in contrast previous determinations with ion-selective microelectrodes using the small cation tetramethylammonium (mol. wt = 74.1) give tortuosities of about 1.6. The results show that proteins as large as bovine serum albumin diffuse through brain extracellular space but are more hindered than smaller molecules. A simple model compared the differences in diffusion properties of bovine serum albumin, dopamine and nitric oxide in brain tissue and discussed the implications for volume transmission of chemical information between cells. The results are also relevant to the behavior of diffusible factors in brain development and the delivery of therapeutic agents.