Sequential measurements of in vitro Newcastle disease virus-induced interferon (IF) synthesis, spontaneous RNA synthesis, and PHA-induced blastic transformation were made in leukocytes derived from ten patients with infectious mononucleosis (IM). The median leukocyte IF production on the day of diagnosis was found to be significantly depressed, 35 international units (IU) IF per 2 x 106 leukocytes, in comparison to a median titer of 463 IU after recovery (p < 0.002) or to a median of 406 IU found in 52 cultures from normal healthy individuals. No spontaneous production of leukocyte IF or circulating serum IF was noted. An increased rate of spontaneous leukocyte RNA synthesis during the first 2 hr of culture was noted in nine of the ten subjects (p < 0.001). Four of the ten patients showed a markedly defective response to PHA after 96-144 hr in culture. A suggestive, but not statistically significant, correlation was noted between increased spontaneous RNA synthesis, defective late PHA transformation, and defective virus-induced IF production. All defects persisted during the 1-wk study period and were resolved after recovery. We conclude that major temporary alterations occur in several parameters of leukocyte function in IM.