PURPOSE. To map the cellular and subcellular distribution of DNase IIβ activity in the mouse lens. METHODS. DNase IIβ-specific activity was determined by assaying lens lysates prepared from wild-type or DNase IIβ-null mice. Regional nuclease activity was determined by microdissection of lens samples or a tissue-imprinting assay. Subcellular distribution was determined by density-gradient ultracentrifugation. RESULTS. DNase IIβ transcripts increased 200-fold in abundance during fiber cell formation, and DNase IIβ activity accounted for approximately 50% of the acid nuclease activity in the cortical fiber cells. Examination of lenses from DNase IIβ-null mice confirmed that the enzyme was required for denucleation. In wild-type lenses, nuclei were TUNEL positive before denucleation, indicating that 3′-OH DNA ends had accumulated. However, DNase IIβ-mediated scission generates 3′-PO4- DNA ends only. This paradoxical finding was explained by the presence of phosphatases that converted the 3′-PO4- ends produced by DNase IIβ into 3′-OH ends. DNase IIβ activity was strongest early in differentiation, where it was associated with the lysosomal fraction. Later, an increasing proportion of DNase IIβ activity was found in the cytosol. CONCLUSIONS. DNase IIβ activity correlated with and was necessary for fiber denucleation and was most likely contained initially within fiber cell lysosomes before release into the cytoplasm.