We investigated the binding of a fluorescent diltiazem analogue (3R,4S)-cis-1-[2-[[3-[[3-[4,4-difluoro-3a,4-dihydro-5,7-dimethyl-4-bo ra-3a,4a-diaza-s-indacen-3-yl]propionyl]amino]propyl]amin o]ethy]-1,3,4,5-tetrahydro-3-hydroxy-4-(4-methoxyphenyl)-6-(triflu oromethyl)-2H-1-benzazepin-2-one (DMBODIPY-BAZ) to L-type Ca2+ channels in the presence of different 1,4-dihydropyridines (DHPs) by using fluorescence resonance energy transfer (FRET) [Brauns, T., Cai, Z.-W., Kimball, S. D., Kang, H.-C., Haugland, R. P., Berger, W., Berjukov, S., Hering, S., Glossmann, H., & Striessnig, J. (1995) Biochemistry 34, 3461]. When channels are occupied with DMBODIPY-BAZ, a rapid fluorescence change occurred upon addition of different DHPs. The direction of this intensity modulation was found to be only dependent on the chemical composition of the dihydropyridine employed. DHPs containing a nitro group decreased, whereas others (e.g., isradipine) enhanced the fluorescence signal. In addition, all DHPs markedly decreased the association rate constant for DMBODIPY-BAZ without affecting equilibrium binding. Both observations together are best explained by a steric model where the DHP binding site is located in close proximity to the accession pathway of DMBODIPY-BAZ.