In rats given 700 r of total-body x-irradiation, ribonuclease specific activity in spleen homogenates increased twofold, as compared to control samples, 16 hours postirradiation, and returned to the control level 64 hours postirradiation. The distribution of total ribonuclease activity in subcellular fractions of control spleen homogenates was 16.0, 35.7, 20.4, and 28.4% for the nuclear, mitochondrial, microsomal, and supernatant fractions, respectively. Sixteen hours after irradiation, the distribution had changed to 8.7, 29.3, 17.6, and 44.8% for the four fractions, respectively, and at 64 hours it was 4.8, 15.0, 14.1, and 66.0%. The total organ ribonuclease activity 16 hours postirradiation increased 54% over the control level, representing a clear case of enzyme activation at this time period. The considerable change in distribution of total ribonuclease that occurred, which resulted in a great loss of enzyme activity by the particulate fractions and a corresponding gain by the supernatant fraction, is discussed in reference to the behavior of other spleen enzymes after irradiation and in terms of the possible occurrence of spleen ribonuclease in lysosomal particles.