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Amoebae of Dictyostelium discoideum show adaptation towards a chemotactic cyclic AMP signal. Within a few seconds of receipt of the signal they are inhibited for a period of 1-2 min from further chemotactic responses to subsequent cyclic AMP signals of similar or smaller magnitude. The site of this adaptation mechanism in the chemotactic transduction pathway was investigated by addition of components of the transduction chain (GTP analogues, myo-inositol-1,4,5-trisphosphate (InsP3) and Ca2+) to permeabilized cells followed by determination of the amount of cyclic GMP formed as a measure of the chemotactic response. This approach was made possible by finding that permeabilization of amoebae with saponin did not uncouple the cell surface cyclic AMP receptors from stimulation of cyclic GMP formation. It was found that InsP3 and Ca2+ were 'downstream' from the adaptation mechanism: they could trigger a cyclic GMP response in cyclic AMP-adapted amoebae but could not themselves induce adaptation. In contrast, GTP gamma S was unable to trigger a cyclic GMP response in cyclic AMP-adapted cells, although it could trigger multiple cyclic GMP responses in non-adapted cells. We deduce that the site of adaptation to cyclic AMP stimulation is at the G-protein involved in this signalling pathway. Moreover, as GTP gamma S was found to be unable to induce adaptation, we conclude that the mechanism of adaptation involves an action of the cyclic AMP receptor on the G-protein that is distinct from its commonly reported action of stimulating G-protein binding of GTP.