We labeled substance P (SP), neurokinin A (NKA), and [Lys0]gastrin-releasing peptide-27 (GRP) with cyanine 3.18 (cy3). Cy3-peptides purified by HPLC were fully active, determined by [Ca2+]i mobilization. Binding was specific because it was abolished by unlabeled peptides and receptor antagonists. Transfected cells yielded a log-fold greater cy3 intensity than control cells by FACS. Confocal microscopy of transfected cells and cultured enteric neurons showed that cy3-SP bound to surface receptors and was internalized. Internalization was observed in living cells by capture of sequential images. Recovery of surface binding sites was monitored by flow cytometry using cy3-SP. Thus, cy3 neuropeptides can be used to isolate and study receptor-bearing cells.