The pre-Boetzinger Complex (PBC) in the rostral ventrolateral medulla contains a kernel involved in respiratory rhythm generation. So far, its respiratory activity has been predominantly analyzed by electrophysiological approaches. Recent advances in fluorescence imaging now allow for the visualization of neuronal population activity in rhythmogenic networks. In the respiratory network mainly voltage-sensitive dyes have been used so far, but their low sensitivity prevents an analysis of activity patterns of single neurons during rhythmogenesis. We now succeeded using more sensitive Ca²⁺ imaging to study respiratory neurons in rhythmically active brainstem slices of neonatal rats. For the visualization of neuronal activity fluo-3 was suited best in terms of neuronal specificity, minimized background fluorescence, and response magnitude. The tissue penetration of fluo-3 was improved by hyperosmolar treatment during dye loading (100 mM mannitol). Rhythmic population activity was imaged with single cell resolution using a sensitive CCD-camera and a 20x objective and it was correlated with extracellularly recorded mass activity of the contralateral PBC. Correlated optical neuronal activity was obvious online in 29% of slices. Deeper located rhythmic neurons became detectable during offline image processing. Based on their activity patterns, 74% of rhythmic neurons were classified as inspiratory-like and 26% as expiratory-like. Our approach is well suited to visualize and correlate the activity of several single cells with respiratory network activity. We demonstrate that neuronal synchronization and possibly even network configurations can be analyzed in a non-invasive approach with single cell resolution and at frame rates currently not reached by most scanning-based imaging techniques.