We have designed and synthesized a biotinylated vasopressin antagonist which is a selective probe for studying the V1a subtype of vasopressin receptor. Initially we synthesized the novel vasopressin analogue d(CH2)5Tyr(Me)2LysNH2(9)AVP (ALVP). Biotinamidocaproate was subsequently coupled to the epsilon-amino group of ALVP to generate the novel biotinylated probe d(CH2)5Tyr(Me)2Lys(N epsilon-biotinamido-caproate)NH2(9)AVP (ALBtnVP). Pharmacological characterization of ALVP and ALBtnVP established that both ligands were high affinity antagonists at V1a receptors, and that both displayed marked V1a/V2 selectivity. The observation that receptor-bound ALBtnVP was bi-functional, and thereby able to bind conjugated derivatives of avidin or streptavidin, allowed ALBtnVP to be utilized as a selective probe for V1a receptors. This strategy allowed the visualization of V1a receptors on the surface of WRK-1 cells and hippocampal neurons, by using streptavidin-gold with electron microscopy and fluorescein-avidin with light microscopy. We conclude that ALBtnVP is a useful probe for V1a receptors.