1 A comparison has been made of the alpha 1-adrenoceptor controlling gluconeogenesis in tubules from rat renal cortex and [3H]-prazosin binding in membranes prepared from the same tissue under physiological conditions. 2 In renal tubules the alpha-adrenoceptor agonists, oxymetazoline, (--)-noradrenaline, (--)-alpha-methylnoradrenaline and (--)-phenylephrine, stimulated gluconeogenesis from pyruvate. Oxymetazoline was the most potent agonist (EC50 15.7 nM) but produced only 61% of the maximum response elicited by (--)-noradrenaline. 3 The alpha-adrenoceptor antagonists, BE2254, prazosin, indoramin and phentolamine inhibited (--)-noradrenaline-mediated increases in gluconeogenesis. The alpha 1-adrenoceptor selective compounds, BE2254 and prazosin, were the most effective antagonists with KB values of 0.74 and 1.47 nM respectively. 4 [3H]-prazosin binding to membranes prepared from rat renal cortex in physiological saline at 37 degrees C was best described by a two site model. High affinity, but not low affinity sites had characteristics consistent with alpha-adrenoceptors. 5 High affinity [3H]-prazosin binding could be completely displaced by the alpha-adrenoceptor agonists, oxymetazoline, (--)-noradrenaline, (--)-phenylephrine, and (--)-alpha-methylnoradrenaline. Slope factors for the displacement curves were all significantly less than unity. The concentrations of agonists required to displace [3H]-prazosin binding were markedly higher than those required to stimulate gluconeogenesis. 6 High-affinity [3H]-prazosin binding was also displaced by the alpha-adrenoceptor antagonists, prazosin, BE2254, phentolamine and indoramin. Slope factors for the displacement curves were close to unity. Ki values calculated from the binding experiments were very similar to KB values obtained in the gluconeogenesis studies. These results suggest that in rat renal cortex the alpha 1-adrenoceptor labelled by [3H]-prazosin is probably that which stimulates gluconeogenesis.