Phenotypic identification of pre-Bötzinger complex (pre-BötC) neurons is vital to the delineation of endogenous cellular properties involved in respiratory rhythm generation and neuromodulation of breathing. A subpopulation of pre-BötC neurons endowed with intrinsic rhythmicity is proposed as the kernel for inspiratory rhythm generation in vitro. In this study, the pre-BötC "island" preparation was excised from medullary slices and reduced to its heterogeneous cellular constituents using primary cell culture techniques. Cultured neurons were labeled with tetramethylrhodamine conjugated to Substance P (TMR-SP) to identify individual NK-1R expressing neurons. Data from this study provide initial evidence that the pre-BötC neurons living in culture remain functionally capable of binding and internalizing the fluorescently-tagged ligand and TMR-SP tagging is a useful experimental tool that reliably identifies the NK-1R phenotype.