A study was undertaken to determine the subcellular location of DNase-I, a major acid deoxyribonuclease in Drosophila melanogaster. Embryonic tissue used in these experiments was derived from a wild type strain and from a strain homozygous at the DNase-l locus (3-61.8) for the null activity allele, DNase-lnl24. The majority of total acid DNase and DNase-l activity is found in the small particulate fraction of tissue homogenates fractionated by differential centrifugation. The activity exhibits latency in these extracts indicating that it is membrane delimited. DNase-I activity also co-equilibrates in sucrose density gradients with acid phosphatase activity which, in D. melanogaster, is known to be lysosomal. These results suggest that DNase-l is localized largely within the Iysosomes of embryonic tissue. Functional implications of a lysosomal location for DNase-l are discussed.