An iodoazido[125I]prazosin analogue was employed to photoaffinity label alpha 1-adrenergic receptors in rat liver plasma membranes. Labeled proteins were separated by gradient polyacrylamide gel electrophoresis in sodium dodecyl sulfate, and (-)-epinephrine displacement of [3H]prazosin binding was concurrently measured in the presence or absence of guanosine 5'-O-(gamma-thiotriphosphate) (GTP[gamma S]). Inclusion of EGTA and/or proteinase inhibitors during membrane preparation and incubation increased the effect of GTP[gamma S] on alpha 1-adrenergic agonist binding and this could be correlated with increased concentrations of a 78 kDa photoaffinity labeled protein. In contrast, omission of EGTA or addition of exogenous Ca2+ diminished or abolished the effect of GTP[gamma S] on binding and caused loss of the 78 kDa form and the appearance of lower molecular weight labeled proteins. Age-dependent differences in GTP[gamma S] effects on alpha 1-adrenergic agonist binding were abolished when membranes were prepared and incubated in the presence of EGTA and proteinase inhibitors. However, the 78 kDa photoaffinity labeled protein observed in adult rats (over 225 g body weight) was not apparent in membranes from younger rats (50-75 g), even when the membranes were prepared and incubated in the presence of EGTA and proteinase inhibitors. Instead, a 68 kDa species was the major labeled protein. These data suggest that GTP effects on alpha 1-adrenergic agonist binding in rat liver membranes require the presence of either a 68 or 78 kDa alpha 1-adrenergic binding protein. Failure to inhibit proteolysis in the membranes leads to the generation of lower-molecular-weight binding proteins and the loss of GTP effects on alpha 1-adrenergic agonist binding, although [3H]prazosin binding characteristics are not changed. It is suggested that either the proteolyzed forms of the alpha 1-adrenergic receptor are unable to couple to a putative guanine nucleotide-binding regulatory protein, or that such a protein is concurrently proteolyzed and is thus unable to couple to the receptor.