Fluorescein isothiocyanate isomer I (FITC-I) conjugates of 2-(3,4-dichlorophenyl)-N-methyl-N-[1-(3- or 4-aminophenyl)-2-(1-pyrrolidinyl)ethyl]acetamide (10 and 14) were prepared either without or with an intervening mono-, di-, or tetraglycyl linker. The 3-substituted fluorescent probes (2-5) were found to retain potent agonist activity in smooth muscle preparations as well as high κ receptor affinity and selectivity in receptor binding assays. The 4-substituted series (6-9) were substantially less potent than the corresponding 3-substituted compounds. Flow cytometric analysis demonstrated high levels of direct κ-specific staining of mouse microglial cells by the fluorescent probe 5 containing a tetraglycyl linker, as indicated by a 41% decrease in percent cells positively labeled and a 61% decrease in mean fluorescence intensity in the presence of the κ-selective antagonist, norbinaltorphimine (norBNI). In similar studies, the probe 2 without a linker exhibited only nonspecific binding. This is the first report of direct, selective staining of κ opioid receptors by a fluorescent nonpeptide opioid ligand. The results of the present study illustrate the importance of introducing hydrophilic linkers to reduce nonspecific binding of fluorescent probes for opioid receptors.