The degree of responsiveness of lymphoid cells to activation by mitogens and antigens is commonly assessed in vitro by measuring radioactive DNA precursor incorporation. Several lines of evidence indicate that artifacts affect the results and that these measurements may not be an accurate reflection of cell activation. Cultures of blastogenically activated lymphocytes contain soluble, noncytotoxic factors that inhibit the incorporation of radioactive nucleosides into DNA by dividing cells without affecting their rate of DNA synthesis. Inhibitors were found in the serum component of the medium and in the bacterial homogenates used to activate the cells, and they were produced by the activated cells. Inhibitor activity in serum has properties expected of a nucleoside such as thymidine, including a molecular weight of less than 10(3). The inhibitor activity present in some bacterial homogenates and that produced by activated cells enzymically degrade labeled DNA precursors, thereby preventing their availability for incorporation. Other bacterial preparations contain DNA precursors, which compete with labeled nucleosides for incorporation, and additional low-molecular-weight inhibitor is produced when the preparations are incubated. Preparations of various bacteria differ greatly with regard to the potency of their inhibitor activity. In some cases incorporation of label in activated cultures is reduced to background levels. Inhibition by these substances leads to erroneous conclusions regarding the proliferative activity of cultured lymphocytes, since the amount of label incorporated does not accurately indicate the true rate of DNA synthesis of the cells.