The structural properties of brain extracellular space (ECS) are summarised by the tortuosity (lambda) and the volume fraction (alpha). To determine if these two parameters were independent, we varied the size of the ECS by changing the NaCl content to alter osmolality of bathing media for rat cortical slices. Values of lambda and alpha were extracted from diffusion measurements using the real-time ionophoretic method with tetramethylammonium (TMA+). In normal medium (305 mosmol kg(-1)), the average value of lambda was 1.69 and of alpha was 0.24. Reducing osmolality to 150 mosmol kg(-1), increased lambda to 1.86 and decreased alpha to 0.12. Increasing osmolality to 350 mosmol kg(-1), reduced lambda to about 1.67 where it remained unchanged even when osmolality increased further to 500 mosmol kg(-1). In contrast, alpha increased steadily to 0.42 as osmolality increased. Comparison with previously published experiments employing 3000 M(r) dextran to measure lambda, showed the same behaviour as for TMA+, including the same constant lambda in hypertonic media but with a steeper slope in the hypotonic solutions. These data show that lambda and alpha behave differently as the ECS geometry varies. When alpha decreases, lambda increases but when alpha increases, lambda rapidly attains a constant value. A previous model allowing cellular shape to alter during osmotic challenge can account qualitatively for the plateau behaviour of lambda.