This study provides a critical examination of protein labeling with Cy3, Cy5, and other Cy dyes. Two alternate situations were tested. (i) Antibodies were covalently labeled with Cy dye succinimidyl ester at various fluorophore/protein ratios and the fluorescence of the labeled antibodies was compared to that of free Cy dye. (ii) Fluorescent biotin derivatives were synthesized by derivatizing ethylenediamine with one biotin and one Cy3 (or Cy5) residue. The fluorescence properties of these biotin-Cy dye conjugates were examined at all ligand/(strept)avidin ratios (0 </= n </= 4). The results showed an astounding discrepancy between Cy3 and Cy5: Cy3-labeled antibodies fluoresced very well, even at high Cy3/protein ratios, and the same applied to (strept)avidin with up to four bound biotin-Cy3 conjugates. In contrast, antibodies with six covalently bound Cy5 labels (obtained with the recommended procedure) were almost nonfluorescent, only at 2-3 Cy5 labels/IgG some moderate fluorescence was obtained. By analogy, the biotin-Cy3 conjugate fluoresced intensely, even at high ligand/avidin ratio, in contrast to the weakly fluorescing biotin-Cy5 conjugate. Three mechanisms are responsible for the discrepancy between Cy3 and Cy5. (i) Attachment of Cy3 to a protein's surface causes an anomalous enhancement in fluorescence (by 2-3-fold) while no enhancement occurs with Cy5. (ii) Mutual quenching of IgG-bound Cy dyes by resonance energy transfer is much more pronounced for Cy5 labels than for Cy3. (iii) In IgG with six bound Cy5 labels, about one-third of the labels adopt a nonfluorescent state which is characterized by a large UV-vis absorption maximum at 600 nm instead of at 650 nm. Cy3.5 was found to mimick the properties of Cy3, while Cy7, and to some extent also Cy5.5, were similar to Cy5. In conclusion the Cy dye series is divided into two groups: Antibodies with multiple Cy3 or Cy3.5 labels yield bright fluorescence while extensive quenching occurs in antibodies labeled with Cy5 and Cy7.