Activation of adenosine A_2A receptors (A_2AR) has been shown to antagonize the function of D₂ dopaminergic regulation of striatal γ‐aminobutyric acid (GABA)‐ergic output and, thus, locomotor activity. Adenosine A_2A receptor immunoreactivity (A_2A‐LI) has been localized to rat striatum by light microscopy by using a previously characterized human A_2AR monoclonal antibody. In this study, we evaluated the localization of A_2A‐LI and its colocalization with GABA immunoreactivity (GABA‐LI) in dorsolateral rat striatum by immunoelectron microscopy to further characterize the potential mechanism of purinergic control of striatal output. Ultrastructural analysis demonstrated A_2A‐LI associated with the plasma membrane and cytoplasmic membranous structures of striatal neurons. A_2A‐LI was prevalent in dendrites and dendritic spines (∼70% of total A_2A‐profiles counted) and less prevalent in axons and axon terminals (23%), soma (3%), and glia (3%). Cellular elements exhibiting both A_2A‐LI and GABA‐LI comprised 23% of the total profiles counted; colabeling was most common in dendrites. A_2A‐LI was observed primarily at asymmetric synapses (n = 70) (both pre‐ and postsynaptically but predominantly in the postsynaptic element) and less frequently at symmetric synapses (n = 17). Of the 714 A_2A‐immunoreactive profiles examined, 37% were apposed to GABA‐labeled profiles. The most common appositions were A_2A‐labeled dendrites apposed to GABA‐immunoreactive dendrites (n = 132), axon terminals (n = 28), and somata (n = 22) and A_2A‐labeled axons apposed to GABA‐labeled dendrites (n = 58), axon terminals (n = 14), and somata (n = 9). Our findings suggest that adenosine may play an important role in modulating excitatory input to striatal neurons and that A_2AR may modulate GABAergic signaling at several cellular sites within the rat striatum. J. Comp. Neurol. 431:331–346, 2001. © 2001 Wiley‐Liss, Inc.