Photopharmacology is an emerging approach that allows the spatiotemporal control of receptor function using photodrugs and offers superior tissue specificity and reduced off-target effects. Previously, we successfully demonstrated the blockade the adenosine A2A receptor (A2AR) in mouse brain using MRS7145, a photocaged derivative of the selective A2AR antagonist SCH442416.1 Here, we explore the use of bioluminescence resonance energy transfer (BRET) to photouncage MRS7145 in vitro and in vivo. First, we demonstrated the effective release of SCH442416 by BRET uncaging or bioluminolysis of MRS7145 in HEK-293 expressing A2AR tagged with nanoluciferase (NL) at the N-terminus (i.e., A2ARNL). Bioluminolysis-mediated A2AR blockade was monitored by cAMP accumulation determinations after CGS21680 activation. Next, we expressed A2ARNL in the mouse brain using an adeno-associated virus (AAV). Correct expression of A2ARNL in the mouse striatum was confirmed by luminescence recordings and immunohistochemical detection of A2ARNL. The photochemical properties and pharmacokinetics of several NL substrates for in vivo use in behaving mice were characterized. Finally, the bioluminolysis activation of MRS7145 in living animals is tested through several behavioural tests, namely locomotor activity and anticataleptic activity. Our method shows potential to improve animal behaviour studies by eliminating the requirement of restraining cables or implantation of optical fibres.1. Taura, J. et al. Remote control of movement disorders using a photoactive adenosine A2A receptor antagonist. Journal of Controlled Release 283, 135–142 (2018).