It has been suggested there is a decreased renal responsiveness to vasopressin following spaceflight and that this may be the mechanism for the increased urine flow that is observed following return to normal gravity. In the present study, we have therefore measured vasopressin receptor expression and activity in kidneys taken from rats 1 and 14 days following spaceflight of 15 days duration. Measurements of renal vasopressin V₂ and V_1a receptor mRNA expression by quantitative RT-PCR demonstrated little difference at either 1 day or at 14 days following return from space. Evaluation of ³H-labeled arginine vasopressin binding to membranes prepared from kidneys indicated that the majority of the vasopressin receptors were V₂ receptors. Furthermore, the data suggested that binding to vasopressin V₂ or V_1a receptors was unaltered at 1 day and 14 days following spaceflight. Similarly, the ability of vasopressin to stimulate adenylate cyclase suggested no change in vasopressin V₂ receptor activity in these animals. These data suggest that, whatever changes in fluid and electrolyte metabolism are observed following spaceflight, they are not mediated by changes in vasopressin receptor number or vasopressin-induced stimulation of adenylate cyclase.