A key question in receptor signaling is how specificity is realized, particularly when different receptors trigger the same biochemical pathway(s). A notable case is the two β‐adrenergic receptor (β‐AR) subtypes, β 1 and β 2 , in cardiomyocytes. They are both coupled to stimulatory G s proteins, mediate an increase in cyclic adenosine monophosphate (cAMP), and stimulate cardiac contractility; however, other effects, such as changes in gene transcription leading to cardiac hypertrophy, are prominent only for β 1 ‐AR but not for β 2 -AR. Here, we employ highly sensitive fluorescence spectroscopy approaches, in combination with a fluorescent β‐AR antagonist, to determine the presence and dynamics of the endogenous receptors on the outer plasma membrane as well as on the T-tubular network of intact adult cardiomyocytes. These techniques allow us to visualize that the β 2 ‐AR is confined to and diffuses within the T-tubular network, as opposed to the β 1 ‐AR, which is found to diffuse both on the outer plasma membrane as well as on the T-tubules. Upon overexpression of the β 2 ‐AR, this compartmentalization is lost, and the receptors are also seen on the cell surface. Such receptor segregation depends on the development of the T-tubular network in adult cardiomyocytes since both the cardiomyoblast cell line H9c2 and the cardiomyocyte-differentiated human-induced pluripotent stem cells express the β 2 ‐AR on the outer plasma membrane. These data support the notion that specific cell surface targeting of receptor subtypes can be the basis for distinct signaling and functional effects.