The role of adenosine in the modulation of respiration-related neurons was examined using an in vitro brainstem-spinal cord preparation from neonatal rats (0-4 d old). Respiratory activity was recorded from the C4 or C5 ventral roots by suction electrodes and from inspiratory related neurons (I neurons) in the rostral ventrolateral medulla by microelectrodes. The following substances were added to the preparation superfusate, and their effect was evaluated: the adenosine A1 receptor agonist N6-(2-phenylisopropyl)adenosine, R(-)isomer (R-PIA), the adenosine uptake blocker dipyridamole, the adenosine receptor antagonist theophylline, and the specific A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). R-PIA and dipyridamole decreased the activity of I neurons and the C4 respiratory burst rate. Furthermore, these compounds induced a significantly more irregular respiratory rate in three-quarters of preparations from the youngest animals (<48 h old) compared with that of controls. Theophylline or DPCPX reversed the effects of both R-PIA and dipyridamole on respiratory rate, regularity of respiratory rate, inspiratory time, amplitude, and intra-burst frequency of I neurons. Thus, adenosine depresses both the I neurons in the rostral ventrolateral medulla and the respiratory motor output. This depression of I neurons and respiratory rate can be abolished by theophylline primarily through a blockade of medullary adenosine A1 receptors. An age-dependent correlation of the effects of R-PIA and dipyridamole, with a more pronounced decrease in respiratory activity in preparations from younger animals, indicates that adenosinergic modulation of medullary respiration-related neurons changes during the first days of postnatal life.