In the respiratory network of mice, we characterized with the whole cell patch-clamp technique pacemaker properties in neurons discharging in phase with inspiration. The respiratory network was isolated in a transverse brain stem slice containing the pre-Bötzinger complex (PBC), the presumed site for respiratory rhythm generation. After blockade of respiratory network activity with 6-cyano-7-nitroquinoxalene-2,3-dione (CNQX), 18 of 52 inspiratory neurons exhibited endogenous pacemaker activity, which was voltage dependent, could be reset by brief current injections and could be entrained by repetitive stimuli. In the pacemaker group (n = 18), eight neurons generated brief bursts (0.43 +/- 0.03 s) at a relatively high frequency (1.05 +/- 0.12 Hz) in CNQX. These bursts resembled the bursts that these neurons generated in the intact network during the interval between two inspiratory bursts. Cadmium (200 microM) altered but did not eliminate this bursting activity, while 0.5 microM tetrodotoxin suppressed bursting activity. Another set of pacemaker neurons (10 of 18) generated in CNQX longer bursts (1.57 +/- 0.07 s) at a lower frequency (0.35 +/- 0.01 Hz). These bursts resembled the inspiratory bursts generated in the intact network in phase with the population activity. This bursting activity was blocked by 50-100 microM cadmium or 0.5 microM tetrodotoxin. We conclude that the respiratory neural network contains pacemaker neurons with two types of bursting properties. The two types of pacemaker activities might have different functions within the respiratory network.