alpha 2-Adrenergic receptors in calf retina membranes can be specifically labeled with the tritiated antagonist 3H-RX 781094. Saturation binding occurs to a single class of noncooperative sites. The number of sites amounts to 1070 +/- 243 and 935 +/- 178 fmol/mg of protein, and the equilibrium dissociation constants equal 1.8 +/- 0.4 and 3.8 +/- 0.3 nM at 25 degrees and 37 degrees, respectively. Binding is rapid, equilibrium being reached within 5 min, and is reversible. At both temperatures, (-)-epinephrine competition binding curves are shallow in the presence of magnesium ions. The curves, obtained for incubation periods varying between 5 and 60 min, are superimposable at 37 degrees. Computer-assisted analysis indicates that approximately 75% of the receptors (RH sites) display high agonist affinity for (-)-epinephrine as well as for the other agonists tested: (-)-norepinephrine, clonidine, and UK 14304. However, the (-)-epinephrine competition curves display a time-dependent leftward shift at 25 degrees. This can be attributed to an increase in agonist affinity for the RH sites. Addition of 0.1 mM Gpp(NH)p causes a marked steepening and rightward shift of the curves, at both 25 and 37 degrees. These curves are superimposable for all of the incubation times tested. The nonequilibrium of agonist competition binding at 25 degrees can be attributed to slow dissociation of the agonist (i.e., tight binding) when the receptor is coupled to the regulatory component Ni. This dissociation rate can be measured by preincubation of the membranes with 10 microM (-)-epinephrine, followed by extensive washing and incubation with 3H-RX 781094 for increasing lengths of time. The first order rate of agonist dissociation (i.e., receptor recovery) is appreciably faster at 37 degrees than at 25 degrees: i.e., 0.029 min-1 and 0.0044 min-1, respectively. These findings are confirmed by kinetic experiments using the radiolabeled agonist 3H-UK 14304. Slow agonist dissociating kinetics may prevent the correct evaluation of the agonist binding parameters by computerized analysis of competition binding curves when the incubation time is too short, especially at low temperature.