Chemical substrates, central sites and central mechanisms underlying the regulation of breathing in lower vertebrates have not been well characterized. The present study was undertaken to determine the effect of pH changes and cholinergic agents on the central control of respiration in the cane toad, Bufo marinus. Adult toads were anesthetized, catheterized and unidirectionally ventilated before exposing the brainstem. An airtight buccal cannula was also inserted through the tympanum to record buccal pressure. The animal was decerebrated, anesthetic removed and the responses to pH changes of solutions bathing the ventral surface of the medulla (VSM) were tested by superfusing the VSM with mock cerebrospinal fluid (mCSF) of pH 7.8-normal, 7.2-acidic and 8.4-basic. The acidic solution increased respiratory activity, the basic solution decreased activity and the normal solution had no effect. In addition, cholinergeric agents (acetylcholine-ACh, physostigmine-Phy, nicotine-Nic, and atropine-Atr) dissolved in mCSF were applied bilaterally onto the VSM using filter paper pledgets. ACh, Phy and Nic increased episodic breathing frequency by 14.3+/-9.7, 9.4+/-5.4 and 29.1+/-11.8 %, respectively, whereas, Atr caused a decrease (-26.6+/-16.6%). These agents had no effect on blood pressure. It is therefore, concluded that the VSM is pH sensitive and a cholinergic mechanism is involved in the central modulation of respiration in Bufo.