An immunofluorescent technique that is more sensitive than radioligand binding was developed in order to detect opioid receptors expressed on leukocytes. The current study was designed to optimize the method for fluorescently labeling kappa opioid receptors. For these experiments, the opioid antagonist naltrexamine was conjugated to either fluorescein (FITC-NTXamine) or biotin (biotin-NTXamine). One-step, two-step, and three-step protocols were compared to determine which procedure resulted in optimal staining of the kappa opioid receptor expressed on intact, unfixed R1E/TL8x.1.OUAr.1(R1EGO) cells, a thymoma known to express kappa opioid receptors. The one-step method involved incubating cells with FITC-NTXamine, and the fluorescein intensity was measured by flow cytometry. In the two-step method, cells were incubated with biotin-NTXamine, followed by extravidin-conjugated phycoerythrin, and the phycoerythrin fluorescence was measured. Finally, in the three-step protocol, cells were incubated with FITC-NTXamine, followed by biotin-conjugated anti-fluorescein IgG, then extravidin-phycoerythrin. The one-step protocol stained the cells, but the signal was not diminished in the presence of opioid competitors. The two-step approach did not stain cells significantly above background levels. Only the three-step approach yielded staining that was displaced by the kappa-selective antagonist nor-binaltorphimine. Thus, the addition of a secondary biotinylated antibody, resulting in the amplification of binding, which was detected using phycoerythrin as a fluorophore, was required to detect low levels of opioid receptor expression on leukocytes.