A method is described for the preparation of ;free' and ;synaptosomal' brain mitochondria from fractions of guinea-pig cerebral cortex respectively depleted and enriched in synaptosomes. Both preparations of mitochondria have a low membrane H(+) conductance, a high capacity to phosphorylate ADP, and a capacity to accumulate Ca(2+) at rates limited by the activity of the respiratory chain. Ca(2+) transport by ;free' brain mitochondria is compared with that of heart mitochondria. The Ca(2+) conductance of ;free' brain mitochondria was at least 20 times that for rat heart mitochondria. Ca(2+) uptake by brain mitochondria increased the pH gradient and decreased membrane potential, whereas little change occurred during the much slower uptake by heart mitochondria. In the presence of ionophore A23187, dissipative Ca(2+) cycling decreased the H(+) electrochemical potential gradient of brain mitochondria from 190 to 60mV, but caused only a slight decrease with heart mitochondria, although the ionophore lowered the pH gradient and increased membrane potential. The Ca(2+) conductance of ;free' brain mitochondria is distinctive in showing a hyperbolic dependency on free Ca(2+) concentration. In the presence of Ruthenium Red, a rapid Na(+)-dependent Ca(2+) efflux occurs. The H(+) electrochemical potential gradient is maintained during this efflux, and membrane potential increases, with both ;free' brain and heart mitochondria. The Na(+) requirement for Ca(2+) efflux appears not to be related to the high Na(+)/H(+) exchange activity but may represent a direct exchange of Na(+) for Ca(2+).