The mitochondrial probe 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyan ine iodide (JC-1) not only identifies mitochondria exhibiting low membrane potentials by the emission of green fluorescence (range, 510-520 nm) but also differentiates these from mitochondria exhibiting relatively high membrane potentials. This discrimination occurs because JC-1 forms aggregates at high membrane potentials. These J-aggregates emit a bright red-orange fluorescence at 590 nm. In this study, JC-1 was combined with the classical dead cell stain, propidium iodide (PI), to identify a spectrum of functional sperm along with degenerate sperm. Flow cytometric analysis of bull sperm showed that the aggregate:monomer ratio differed among bulls before cryopreservation (P < 0.001) but not afterwards (P > 0.05). The effects of stain equilibration time, sperm concentration, and live:dead ratios were examined. The addition of SYBR-14 to the JC-1 and PI combination enhanced the distinction between the red PI-stained and red-orange JC-1-stained populations. This discrimination between J-aggregates and the PI-stained sperm was affected by sperm concentration. These studies show that JC-1 can be useful in monitoring mitochondrial function in bovine sperm.