Rhodamine dyes in aqueous solution form non-fluorescent dimers with a plane-to-plane stacking geometry (H-dimers). The self-quenching properties of these dimers have been exploited to probe the conformation and dynamics of proteins using a variety of fluorescence approaches that require the interpretation of fluorescence intensities, lifetimes and fluctuations. Here, we report on a systematic study of the photophysical properties of three rhodamine dyes (tetramethylrhodamine, Alexa 488 and Alexa 546) covalently bound to the E. coli sliding clamp (β clamp) with emphasis on the properties of the H-dimers that form when the dimeric protein is labeled with one dye at each side of the dimer interface. Overall, results are consistent with an equilibrium between non-emissive dimers and unstacked monomers that experience efficient dynamic quenching Protein constructs labeled with tetramethylrhodamine show the characteristic features of H-dimers in their absorption spectra and a c.a. 40-fold quenching of fluorescence intensity. The degree of quenching decreases when samples are labeled with a tetramethylrhodamine derivative bearing a six-carbon linker. H-dimers do not form in samples labeled with Alexa 488 and A546, but fluorescence is still quenched in these samples through a dynamic mechanism. These results should help researchers design and interpret fluorescence experiments that take advantage of the properties of rhodamine dimers in protein research.