We used the patch clamp technique to study the effect of adenosine on the apical 70 pS K channel in the thick ascending limb (TAL) of the rat kidney. Application of 1 µM cyclohexyladenosine (CHA), an adenosine analog, stimulated the apical 70 pS K channel activity and increased NP_o from 0.34 to 0.7. Also, addition of CGS21680, a specific A2a adenosine receptor agonist, mimicked the effect of CHA and increased NP_o from 0.33 to 0.77. The stimulatory effect of CHA and CGS21680 was completely blocked by H89, an inhibitor of protein kinase A (PKA) or by inhibition of adenylate cyclase with SQ22536. This suggests that the stimulatory effect of adenosine analogs is mediated by a PKA-dependent pathway. The effect of adenosine analog was almost absent in the TAL from rats on a K-deficient (KD) diet for 7 days. Application of DDMS, an agent which inhibits cytochrome P450 hydrolase, not only significantly increased the activity of the 70 pS K channel but also restored the stimulatory effect of CHA on the 70 pS K channel in the TAL from rats on a KD diet. Also, the effect of CHA was absent in the presence of 20-hydroxyeicosatetraenoic acid (20-HETE). Inhibition of PKA blocked the stimulatory effect of CHA on the apical 70 pS K channel in the presence of DDMS in the TAL from rats on a KD diet. We conclude that stimulation of adenosine receptor increases the apical 70 pS K channel activity via a PKA-dependent pathway and that the effect of adenosine on the apical 70 pS K channel is suppressed by low K intake. Moreover, the diminished response to adenosine is the result of increase in 20-HETE formation which inhibits the cAMP-dependent pathway in the TAL from rats on a KD diet.