Decreased immune responses have been documented in hyperbilirubinemic patients. This study investigates the effects of intracellular bilirubin accumulation on lymphoproliferative response to phytohemagglutinin A (PHA). Human peripheral blood mononuclear cells (PBMNC) were preincubated with unconjugated bilirubin dissolved in bovine albumin solution at pathological levels seen in clinical hyperbilirubinemia (0–12 mg/dl), washed, and further cultured with PHA. DNA synthesis was measured by [³H]thymidine uptake. Interleukin-2 (IL-2) activity was determined by the CTLL proliferation assay. The amount of intracellular bilirubin and expression of cell surface antigens were analyzed by flow cytometry.In vitro exposure of normal PBMNC to bilirubin resulted in the accumulation of intracellular bilirubin and a decrease in DNA synthesis after PHA stimulation in a time- and dose-dependent manner. Addition of autologous untreated monocytes could not correct the decreased DNA synthesis of bilirubin-treated lymphocytes. IL-2 production by bilirubin-treated PBMNC after PHA stimulation was significantly decreased compared to bilirubin-untreated PBMNC. However, addition of exogenous IL-2 to pretreated PBMNC could not correct the decreased DNA synthesis. Expression of Tac antigen and transferrin receptor on bilirubin-treated lymphocytes after PHA stimulation was not significantly different from bilirubin-untreated cells. These results suggest that decreased PHA-induced T-lymphocyte proliferation following bilirubin-pretreatment may result from impairment of proliferation at a step beyond transferrin receptor expression. These observations may help explain the increased susceptibility to infection often observed in hyperbilirubinemic patients.