The levels of phosphatidylcholine hydroperoxide in serially cultured human fetal diploid fibroblasts at various population doubling levels were determined by high-performance liquid chromatography combined with chemiluminescence detections. This methodology utilizes a mixture of cytochrome c and luminol as post-column hydroperoxide group specific luminescent reagents. The cellular hydroperoxide content increased with age from 0.34 to 27.72 pmol/106 cells. At the end of the cells'in vitro lifespan (51st population doubling level), the hydroperoxide content per 106 cells reached about 80 times the level found in cells of the 20th population doubling level. Supplementation of exogenous α-tocopherol to the culture medium prevented hydroperoxide accumulation, but did not extent the lifespan in vitro. The results indicate that substantial intracellular phospholipid hydroperoxide accumulation occurred in the course of aging of human fetal liploid fibroblasts.