The contribution of pons and dorsal medulla in establishing the pattern of fictive respiration and in mediating the respiratory response to acidification was studied using the isolated brainstem-spinal cord preparation from neonatal mouse. About 40% of ponto-medullary preparations (retaining pons) showed spontaneous, but irregular respiratory-like rhythm. In the other 60%, the elimination of the pons often was followed by the initiation of a respiratory-like rhythm. Medullary preparations, derived from either inactive or rhythmic ponto-medullary preparations, showed a regular respiratory-like rhythm, which was also of a higher frequency and a bigger amplitude than that observed in ponto-medullary preparations. In contrast, ventral medullary preparations, derived from medullary preparations by eliminating the dorsal medulla, showed an irregular rhythm with a reduced amplitude of the integrated inspiratory burst. In ponto-medullary and ventral medullary preparations, acidification of the superfusion medium increased the respiratory frequency, while in medullary preparations, it increased the frequency and reduced the amplitude of the inspiratory burst. Our results suggest that pontine structures influence negatively the rate and depth of the respiratory-like rhythm, while dorsal medullary structures influence positively the depth of the rhythm. They also suggest that the pattern of response to pH supported by the ventral medulla is modified by the input provided from pons and dorsal medulla.