The triazine dye Cibacron Blue F3GA stimulated calcium uptake into an ionomycin-sensitive compartment of washed bovine sperm. Cibacron blue stimulated uptake was not sensitive to nifedipine, diltiazem, or verapamil, but was blocked by transition metals, 1 μM ruthenium red, rotenone, and the protonophore CCCP. Uptake was dependent on the maintenance of energized mitochondria but not on oxidative or substrate level ATP production and was not supported by glycolysis. The cibacron blue-stimulated uptake was judged by these criteria to be mitochondrial. Uptake in the absence of Cibacron blue was 0.046 ± 0.008 and 0.268 ± 0.068 nmol/min/108 cells with 0.1 mM Cibacron blue at 15 μM Ca2+. Half-maximal stimulation occurred at 20-25 μM Cibacron blue. Cibacron blue also allowed efflux of mitochondrial calcium in the presence of external calcium. This efflux would be blocked by ruthenium red, Ni2+, and La3+, but not by Co2+, which effectively blocked Cibacron blue-stimulated influx. Permeabilizing the plasma membrane with filipin indicated that Cibacron blue stimulation of mitochondrial calcium uptake operates directly on the mitochondria. Cibacron blue does not stimulate mitochondrial respiration and thus, calcium uptake was not the result of increased mitochondrial flux.