Human fibroblasts of both fetal and adult origin incorporated [1-14C] acetate primarily into phospholipid acyl groups (70-80% of total radioactivity). When these labeled cells were replated in non-radioactive medium, there was continuous loss of 14C from steroids, triacylglycerols and non-lipid material. In contrast, after some initial loss, cell lines of fetal origin completely retained 14C in cellular phospholipids during continued cell division. Unlike cells of fetal origin, fibroblasts of adult origin continued to lose radioactivity from their phospholipid acyl groups during growth in unlabeled medium. Loss of radioactivity from [1-3H] acetate incorporated into phospholipids of adult cells cannot be attributed to cell death since it was not accompanied by any loss of previously incorporated [ME-14C] thymidine. If cellular phospholipids were labeled with [U-14C] glycerol, both fetal and adult fibroblasts continued to lose radioisotope from the cells during growth in nonradioactive medium. Thus, there is turnover of the phospholipid molecules themselves in fetal human fibroblasts grown in vitro, but their acyl groups are retained within cellular phospholipids. In this respect, fibroblasts of fetal origin resemble established cell lines such as the L fibroblast. Fibroblasts of adult origin do not exhibit this complete conservation of their phospholipid acyl groups.