Renal proximal tubule cells are particularly vulnerable to injury following ischemia and reperfusion due to their marginal blood supply and high metabolic demand. Renal adenosine receptor (AR) modulations preserve renal function following ischemic-reperfusion injury in vivo. Numerous intracellular proteins have been shown to be pivotal in the signal transduction of adenosine-mediated protection in vivo. However, characterization of the expression and function of ARs and intracellular proteins mediating protection in human proximal tubular cells is lacking. Therefore, we studied the ARs in an immortalized human renal proximal tubular cell (HK-2) line to determine if this cell line could function as an in vitro model of AR coupling. Immunoblotting with AR subtype specific antibodies detected all 4 subtypes of ARs (A<sub>1</sub>, A<sub>2a</sub>, A<sub>2b</sub> and A<sub>3</sub>), several isoforms of protein kinase C (α, δ, and Ε and several heterotrimeric G-protein isoforms (G<sub>i</sub>α, G<sub>s</sub>α and G<sub>q</sub>α). The A<sub>1</sub> and A<sub>3</sub> ARs inhibited forskolin- stimulated adenylyl cyclase activity. The A<sub>1</sub> ARs also activated 42/44-kD ERK mitogen-activated protein kinases via G<sub>i</sub>- and tyrosine kinase-dependent pathways. The A<sub>2a</sub> ARs stimulated adenylyl cyclase activity and activated the protein kinase A→CREB pathway. Chronic (48 h) treatment with a nonselective AR antagonist (8-phenyltheophylline) upregulated A<sub>1</sub>, A<sub>2a</sub> ARs and G<sub>i</sub>α. Conversely, chronic stimulation of HK-2 ARs with a nonselective AR agonist (N-ethylcarbamoyladenosine) downregulated all 4 subtypes of ARs and G<sub>s</sub>α. Based on these findings, HK-2 cells are a useful in vitro model to study the signaling cascades of AR-mediated renal protection.