In rat hepatocytes, the role of cAMP and Ca 2+ as secondary messengers in the ureagenic response to stimulation of specific adenosine receptor subtypes was explored. Analyzed receptor subtypes were: A 1, A 2A, A 2B and A 3. Each receptor subtype was stimulated with a specific agonist while blocking all other receptor subtypes with a battery of specific antagonists. For the A 1 and A 3 adenosine receptor subtypes, the secondary messenger was the cytoplasmic Ca 2+ concentration ([Ca 2+] cyt). Accordingly, the A 1 or A 3-mediated increase in [Ca 2+] cyt and in ureagenic activity were both inhibited by chelating Ca 2+ with either EGTA or BAPTA-AM. Also, Gd 3+ blocked both the increase in [Ca 2+] cyt and ureagenesis, suggesting that a Ca 2+ channel may be involved in the response to both A 1 and A 3. A partial effect was observed with the sarcoplasmic reticulum Ca 2+-ATPase inhibitor thapsigargin. The concentration of cyclic AMP ([cAMP]) increased in response to stimulation of either the A 2A or the A 2B adenosine receptor subtypes, while it decreased slightly in response to stimulation of either A 1 or A 3. The stimulation of either the A 2A or A 2B adenosine receptor subtypes resulted in an increase in [cAMP] and an ureagenic response which were not sensitive to EGTA, BAPTA-AM, Gd 3+ or to thapsigargin. In addition, the adenylyl cyclase inhibitor MDL12,330A blocked the ureagenic response to A 2A and A 2B, but not the response to either A 1 or A 3. Our results indicate that in the ureagenic liver response to adenosine, the secondary messenger for both, the A 1 and A 3 adenosine receptor subtypes is [Ca 2+] cyt, while the message from the A 2A and A 2B adenosine receptor subtypes is relayed by [cAMP].