The binding of [35S]GTP gamma S was characterised with autoradiography in rat brain. The binding was saturable, but the rate of dissociation was very slow. Analysis of binding isotherms revealed one class of binding sites with a Kd of 0.8 microM. The specific binding was 98%. Different guanine nucleotides were all able to compete with [35S]GTP gamma S binding. However, no displacement was seen by the ATP-analogue App[NH]p, indicating that [35S]GTP gamma S does not bind to ATP-sites. Autoradiograms showed a highly homogenous distribution of [35S]GTP gamma S binding, in grey as well as in white matter. However, the pattern changed dramatically in the presence of GTP, which, unlike the non-hydrolysable GTP-analogues Gpp[NH]p and GTP gamma S, did not displace [35S]GTP gamma S binding throughout the brain. In white matter areas the binding was potently displaced, while in many grey matter areas, e.g., the striatum, the binding was seen to increase. This GTP-induced increase in [35S]GTP gamma S binding was strongly Mg(2+)-dependent, with an optimum at 10 mM. This, together with the finding that the regional effects of GTP correspond well to previously reported distribution of low Km GTPase, suggest that the levels of binding of [35S]GTP gamma S in the presence of GTP may reflect functional G-protein activity.