The notion that the cell death machinery is utilized during lens organelle degradation is supported by the observation that well characterized apoptotic substrates are cleaved during this process. Here, we test directly the role of executioner caspases (caspase-3, -6, and -7) in fiber cell differentiation. The distribution of mRNA, protein, and enzymatic activity for each caspase was determined in the mouse lens. Transcripts for all three executioner caspases were identified in lens fiber cells by real time RT-PCR, although only caspase-6 and -7 proteins were detected subsequently by Western blot analysis. Endogenous proteolytic activity was noted for caspase-3 but not caspase-6 or -7. We tested the role of executioner caspases in organelle degradation by examining lenses from mice deficient in each caspase. Knock-out lenses appeared grossly normal with the exception of caspase-3-/- lenses, which exhibited marked cataracts at the anterior lens pole. The distribution of lens organelles was mapped by confocal microscopy. There was no significant difference in the size of the lens organelle-free zone (OFZ)1 between wild-type and knock-out lenses. In response to treatment with staurosporine, caspase-3 and -6 (but not caspase-7) enzymatic activities were induced. We generated double knock-out animals to examine the phenotype of lenses deficient in both caspase-3 and -6. Histological examination of such lenses indicated the presence of a properly formed OFZ. Thus, no single executioner caspase (nor a combination of caspase-3 and -6) is required for organelle loss, although caspase-3 activity may be required for other aspects of lens transparency.