Key points The adenosine‐dependent component of functional dilation of small resistance arterioles acts via A2A receptors located on endothelium to activate KATP channels on associated vascular smooth muscle. A2A receptors are Gs‐coupled, hence receptor occupancy should activate cAMP/protein kinase A (PKA) cell signalling pathways However, pre‐exposure to adenosine alters the PKA dependence of the response and renders the vessel insensitive to PKA inhibition. The adenosine pre‐exposure effect is mimicked by pre‐activation of PKA and is specific to adenosine, as the PKA dependence of dilation to isoproterenol (another Gs‐coupled agonist) is not affected by pre‐exposure. Activation of PKA alone does not induce dilation. An additional signalling mechanism, dependent on increased EC Ca2+ via activation of cyclic nucleotide gated channels, is required together with PKA activation to produce A2A‐dependent vasodilation. This novel identification of variability in signalling downstream from a single receptor to produce the same response may reflect a mechanism for integration of key homeostatic responses. Abstract Adenosine (ADO) is an endogenous vasodilatory purine widely recognized to be a significant contributor to functional hyperaemia. Despite this, many aspects of the mechanisms by which ADO induces dilation in small resistance arterioles are not established, or appear contradictory. These include: identification of the primary receptor subtype; its location on endothelial (EC) or vascular smooth muscle cells; whether ADO acts on KATP channels in these resistance vessels; and the contribution of cAMP/protein kinase A (PKA) signalling to the response. In intravital microscopy studies of intact or EC‐denuded skeletal muscle arterioles, we show that ADO acts via A2A receptors located on ECs to produce vasodilation via activation of KATP channels located on vascular smooth muscle cells. Importantly, we found that the signalling pathway involves cAMP as expected, but that a requirement for PKA activation is demonstrable only if the vessel is not pre‐exposed to ADO. That is, PKA‐dependent signalling varies with pre‐exposure to ADO. Further, we show that PKA activation alone is not sufficient to dilate these arterioles; an additional EC calcium‐dependent signalling mechanism is required for vasodilation to ADO. The ability of arterioles in situ to respond to occupancy of a specific receptor by utilizing different cell signalling pathways under different conditions to produce the same response allows the arteriole to respond to key homeostatic requirements using more than a single signalling mechanism. Clearly, this is likely to be physiologically advantageous, but the role for this signalling flexibility in the integrated arteriolar response that underlies functional hyperaemia will require further exploration.