The effect of temperature on the binding of agonists and antagonists to cardiac muscarinic receptor sites was investigated. 2. Compared to 37 degrees C, carbachol-[3H]QNB and atropine-[3H]QNB competition curves were shifted to the left at lower temperatures. This was also observed with other agonists and antagonists. 3. Such an effect of temperature persisted in the N-ethylmaleimide (NEM)-and dithiothreitol (DTT)-treated membranes. 4. Dissociation of the membrane-bound [3H]QNB was higher at 37 degrees C 40% at 60 min) compared to 18 degrees C (approximately 20%). Both carbachol and atropine increased the dissociation at 37 degrees C to a similar maximum (to about 60%). 5. The results show marked temperature-dependent alterations in the cardiac muscarinic receptor sites and suggest that these occur either by direct action of the temperature on receptor conformation or its indirect action via membrane fluidity. The likely significance of these results is discussed.