Breathing movements in mammals start episodically in utero, are continuous at birth, and except for the briefest of pauses, continue without respite. The neural circuits underlying breathing must be wired correctly by birth, and must change on the fly as the lungs and respiratory muscles mature and then age. These circuits must be stable yet responsive to challenges affecting O2, CO2 and pH levels in the body such as exercise, sleep and hypoxia. They must be well coordinated with other movements generating airflow such as speech, and airway reflexes such as cough or sneeze, as well as movements impacting the respiratory muscles and lungs such as locomotion. Longer-lasting disturbances demand adaptive solutions; for example, breathing must be adjusted to accommodate physical changes associated with weight gain and loss, pregnancy or disease. At the core of breathing movements is rhythmicity. Here we propose to determine where and how respiratory rhythm is generated in mammals. In 1991, we proposed two hypotheses: i) The preBotzinger Complex (preBotC) contains the kernel for generating respiratory rhythm, ii) Neurons with pacemaker properties underlie the generation of respiratory rhythm. These hypotheses have been the basis for a large body of work in laboratories worldwide. Recent work suggests two alternative hypotheses: i) The preBotC and preinspiratory (pre-I) neuron population interact to produce respiratory rhythm, ii) A group pacemaker network underlies rhythm generation. This grant proposes experiments that test these hypotheses and reevaluate new hypotheses. We have designed the proposed experiments in such a way that regardless of the their outcome, the data will add substantially and in unique ways to our understanding of neural mechanisms underlying the generation of respiratory rhythm and pattern. Determination of the mechanisms underlying breathing movements is basic to understanding human physiology and the pathophysiology of many diseases. Development of prophylaxis and treatment of such diseases as Sudden Infant Death Syndrome, apnea of prematurity, central alveolar hypoventilation, congenital central hypoventilation syndrome, sleep apnea and other forms of respiratory failure critically depend on such knowledge. Studying a measurable behavior under controlled in vitro conditions permits novel experiments that can reveal important features of the link between synapses/neurons and behavior that may have general applicability in understanding mammalian brain function.