The fluorescence changes accompanying the binding of the fluorescent calcium channel antagonist,felodipine, to transverse tubule membranes from rabbit skeletal muscle have been used to characterize low-affinity binding sites for 1 ,Cdihydropyridine derivatives in these preparations. In competition experiments, felodipine inhibited the high-affinity binding of (+)- [3H]PN200- 1 10 to transverse tubule membranes with an apparent Ki of 5 f 2 nM. Binding of felodipine to additional low-affinity sites resulted in a large, saturable (Kd = 6 k 2 pM) increase in its fluorescence which could be excited either directly (380 nm) or indirectly via energy transfer from membrane protein (290 nm). The observed fluorescence enhancement was competitively inhibited by other 1 ,Cdihydropyridines with inhibition constants of 3-2 1 pM but was unaffected by the structurally unrelated calcium channel antagonists, diltiazem and verapamil, or by Ca2+, Cd2+, andLa3+. Both high- and low-affinity binding sites appear to be localized in the transverse tubular system, since the magnitude of the observed fluorescence enhancement was higher in these membranes than in microsomal preparations and was directly proportional to the density of high-affinity sites for (+)- [3H]PN200- 1 10.Furthermore, both high- and low-affinity sites appear to be conformationally coupled since, over the same concentration range that the fluorescence changes were observed, felodipine accelerated the rate of dissociation of [3H]PN200-1 10 previously bound to its high-affinity sites. Similar behavior has previously been reported for other 1,Cdihydropyridines [Dunn, S. M. J., & Bladen, C. (1991) Biochemistry 30, 5716-57211. These results suggest that skeletal muscle transverse tubule membranes carry both high- and low-affinity binding sites for 1,Cdihydropyridines and that these sites may be important for regulation of calcium channel activity.