Perfusing the isolated carotid bodies of 27 dogs in vivo with heparinized-eserinized blood, equilibrated with room air, 5% O 2, 15% CO 2, 5% O 2 + 15% CO 2, produced: an increase in ventilation during hypoxia of +149%; hypercapnia, +19%; hypoxia + hypercapnia, +235%. Carotid body venous outflow contained 1966 pg/ml ACh (±374) during hypoxia and 2807 pg/ml ACh (± 340) during hypoxia + hypercapnia. ACh was not recovered during room air or hypercapnia. After perfusion with a hemicholinium, HC-3, the ventilation increase declined to + 83 % during hypoxia, and to + 116% during hypoxia + hypercapnia, with no increase during hypercapnia; ACh declined during hypoxia to 1264 pg/ml (±179) and to 1661 pg/ml (±259) during hypoxia + hypercapnia. This study supports the hypothesis that a cholinergic substance is a factor in carotid body chemoreceptor activity.