We readdressed the question of whether or not rat adenohypophyseal vasopressin receptors have a ligand selectivity which is similar to that of the V1 subtype of vasopressin receptors. Vasopressin analogues substituted in positions 7 and 1 were used. By incubating rat anterior pituitary quarters or by perifusing rat isolated anterior pituitary cells the effect of the vasopressin analogues on the release of β-endorphin-like or adrenocorticotropin-like immunoreactive was examined. The replacement of the proline residue in position 7 by sarcosine or N-methyl-alanine did not change the maximum effect reached but increased the EC50 values 20- or 5-fold, respectively, when compared with arginine vasopres sin. This decrease in β-endorphin-releasing activity was no longer observed after additional removal of the α-amino group of cysteine in position 1. Since these substitutions are known to drastically reduce vasopressor activity, these data sugges that the β-endorphin-releasing activity of vasopressin can be dissociated from its V1 receptor activity. Vasopressin ana logues substituted in position 7 and with deaminopenicillamine or β-mercapto-ββ-cyclopentamethylenepropionic acid it position 1 were found to be weak antagonists of the β-endorphin-releasing activity of vasopressin. Since these analogue: are potent antagonists at the V1 receptor, these data suggest that the deaminopenicillamine and, more so, the β-mercapto ββ-cyclopentamethylenepropionic acid residues in position 1 of vasopressin are strong ‘binding elements’ at the V1 vaso pressin receptor but weak ‘binding elements’ at the adenohypophyseal vasopressin receptor. A positive correlation was found between the EC50 values or inhibition constants of the analogues for their effect on β-endorphin release on the on< hand and their potency to displace 3H-arginine vasopressin binding to anterior pituitary membranes on the other hand Therefore, these data support and extend previous suggestions that the structural requirements of the adenohypophysea vasopressin receptor differ from those of the V1 vasopressin receptor. In this sense, the adenohypophyseal vasopressin receptor may represent a novel type of va