Twenty-four hour preincubation at 37 degrees enhances the mitogen-induced DNA synthesis of human lymphocytes. PGE1, given simultaneously with Con A at the start of lymphocyte culture, inhibits the DNA synthesis. After 24 h preincubation of cells, PGE1 fails to decrease the DNA synthesis. Similarly, preincubation abolished the effect of indomethacin increasing DNA synthesis of freshly separated lymphocytes. The intracellular cAMP level of human mononuclear leukocytes rapidly decreases during in vitro incubation at 37 degrees C. PGE1 elevates the intracellular cAMP of freshly separated lymphocytes to 45 times of its starting level. After 24 h preincubation of cells at 37 degrees C, PGE1 elevates cAMP to a lesser extent. This change of PGE1 action may explain the fact that the effect of exogenous PGE1 and endogenous prostaglandins (the production of which can be inhibited by indomethacin) diminishes after incubation of lymphocytes. It is very likely that the change of the effect of prostaglandins produced in lymphocyte cultures and the spontaneous decrease of intracellular cAMP level explains the enhancement of mitogen-induced lymphocyte proliferation after 24 h preincubation at 37 degrees C.