Suppression of peripheral blood lymphocyte phytohemagglutinin-stimulated 3H-thymidine uptake in vitro by serum lipoprotein fractions enriched in the immunoregulatory low density lipoprotein (LDL-In) and by 25-hydroxycholesterol was compared to determine whether the biologically active constituent of LDL-In might be an oxygenated sterol. Dose titrations indicated that 25-hydroxycholesterol was 100 times more potent than LDL-In on the basis of total sterol. Both were similar in that maximal inhibitory activity was evident when they were present in the cultures for at least 24 hr before mitogen stimulation. However, the suppression of 3H-thymidine uptake by 25-hydroxycholesterol required the continuous presence of the sterol, whereas equivalent suppression by LDL-In was irreversible and did not require the presence of the lipoprotein beyond the preincubation period. 25-Hydroxycholesterol-mediated suppression of 3H-thymidine uptake was mitigated by the addition of cholesterol or mevalonic acid to the lymphocyte cultures, consistent with an effect on sterol biosynthesis, whereas, LDL-In mediated suppression was not. Finally, when chloroquine was used to inhibit lymphocyte lysosomal hydrolysis, which is required for the intracellular degradation and release of cholesterol esters from internalized low density lipoprotein, the phenotypic expression of LDL-In mediated suppression was not altered, indicating that this known pathway of regulation of cellular function by low density lipoproteins is not the primary mode of action of LDL-In. These data indicated that the mode of suppression of mitogen-stimulated thymidine uptake by serum lipoprotein fractions enriched in LDL-In differs from that produced by 25-hydroxycholesterol, and that LDL-In suppression does not appear to result from inhibition of intracellular cholesterol biosynthesis.