In order to study the effect of stress on lymphocyte proliferation, SD rats were restrained with four limbs tied on a frame in supine position at room temperature (20 degrees C) for 20 h, and control animals were not disturbed in home cage. The blood was then collected from the heart under light ether anesthesia. The peripheral blood lymphocytes were separated from heparinized whole blood by density gradient (d 1.077) centrifugation, or the serum was obtained after the blood coagulated at 4 degrees C for about 6h. It was found that the blood lymphocyte proliferation induced by Con A was significantly inhibited in the stressed group as compared with the control (P less than 0.01, n = 8, ANOVA). The result was in accordance with our earlier study in which the animals were stressed with electric shock. In the present study, it was also found that the serum of the stressed animals was capable of suppressing Con A-induced lymphocyte proliferation of normal mice (P less than 0.01, n = 8, ANOVA) to a significant extent. Thus the present experiment suggests that there is some substance with suppressive activity on lymphocyte proliferation in the serum of the stressed rats. The serum lost its suppressive activity when it was heated to 100 degrees C (3 min), treated with 60% methanol or incubated with trypsin (64 micrograms/ml), thus suggesting that the suppressive factor(s) most likely is a kind of protein.